1 
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BACTERIOLOGY 


MEDICINE  AND  SURGERY. 

A  PRACTICAL  MANUAL 

FOR 

PHYSICIANS,  HEALTH  OFFICERS  AND  STUDENTS, 


BY 

WM.  HALLOCK  PARK,  M.D., 

ASSOCIATE  PROFESSOR  OF  BACTERIOLOGY  AND  HYGIENE,  UNIVERSITY    AND  BELLE- 
VUE    HOSPITAL    MEDICAL    COLLEGE,    AND    ASSISTANT    DIRECTOR    OF    THE 
RESEARCH    BACTERIOLOGICAL    LABORATORIES    OF    THE    DE- 
PARTMENT OF  HEALTH,   CITY  OF  NEW  YORK. 

ASSISTED  BY 

A.  R.  GUERARD,  M.D., 

ASSISTANT  BACTERIOLOGIST,   DEPARTMENT  OF  HEALTH,  CITY  OF  NEW  YORK. 
ILLUSTRATED  WITH  87   ENGRAVINGS  AND  2  COLORED   PLATES. 


LEA  BROTHERS  &  CO., 
NEW   YORK   AND  PHILADELPHIA. 


BIOLOGY 

LIBRARY 

G 


Entered  according  to  the  Act  of  Congress,  in  the  year  1899,  by 

LEA  BROTHERS  &  CO., 
In  the  office  of  the  Librarian  of  Congress.     All  rights  reserved. 


DORNAN,  PRINTER. 


[ 


PREFACE. 


IN  the  following  pages  the  attempt  has  been  made 
to  group  together  those  facts  in  Bacteriology  which 
will  constitute  a  sufficient  text-book  for  the  student  and 
which  are  of  direct  practical  value  to  the  physician 
and  health  officer.  Laboratory  technique  is  given  in 
its  essentials  and  to  such  an  extent  as  is  necessary  to 
make  bacteriological  methods  plain  to  the  physician, 
to  guide  him  in  making  the  simple  examinations  pos- 
sible in  his  office,  and  to  show  him  under  what 
conditions  he  can  obtain  diagnostic  or  other  help  from 
bacteriological  examinations  in  laboratories.  The  phy- 
sician can  readily  understand  and  apply  the  essentials 
of  bacteriology,  but  the  actual  carrying  out  of  the 
more  difficult  examinations  should  be  left  to  the 
trained  bacteriologist. 

Such  subjects  as  the  chemical  changes  produced  by 
bacteria,  infection,  immunity,  the  nature  and  use  of 
protective  serums,  and  the  diagnostic  value  of  bacte- 
riological cultures,  are  particularly  emphasized,  since 
knowledge  of  such  subjects  is  of  special  importance  to 

91547 


iv  PREFACE. 

the  practising  physician,  in  that  it  enables  him-  to  ob- 
tain an  intelligent  grasp  of  the  nature  of  the  infectious 
diseases. 

The  methods  used  in  the  laboratory  for  the  isolation 
and  identification  of  the  typhoid,  tubercle,  and  diph- 
theria bacilli  have  been  given  with  especial  fulness,  as 
bacteriological  examinations  of  the  discharges  of  per- 
sons suspected  to  have  typhoid  fever,  tuberculosis,  or 
diphtheria  are  now  generally  made  for  these  bacteria 
in  the  laboratories  of  the  health  departments  of  even 
the  smaller  cities,  because  of  the  manifest  importance 
to  the  public  of  knowing  where  such  sources  of  infec- 
tion exist. 

In  preparing  this  book  the  best  works  have  been 
freely  consulted.  Of  these,  those  of  Fliigge  and 
Sternberg,  on  General  Bacteriology,  and  those  of 
Abbott  and  Mallory  and  Wright,  on  Technique,  should 
perhaps  be  especially  mentioned. 

My  sincere  thanks  are  due  to  Dr.  Hermann  M. 
Biggs,  the  Director  of  the  Bacteriological  Laboratory, 
and  to  my  colleagues  in  it,  who  have  so  freely  furnished 
me  with  the  results  of  their  original  investigations.  I 
wish  also  to  especially  acknowledge  my  indebtedness 
to  Dr.  A.  R.  Guerard,  who  has  given  me  invaluable 
aid  in  the  preparation  of  the  book.  The  illustrations, 
with  the  exception  of  those  on  malaria  and  cholera, 
for  which  I  am  indebted  to  Drs.  Welch  and  Dunham, 


PEE  FA  CE. 


are  almost  entirely  from  photographs  taken  from 
cover-glass  preparations  and  cultures  by  Dr.  Edward 
R.  Learning,  Instructor  in  Photography  in  the  Medical 
Department  of  Columbia  University. 


NEW  YORK,  November, 


CONTENTS. 


INTRODUCTION. 

PAGE 

Historical  Sketch  of  the  Development  of  Bacteriology    .         .       17 


CHAPTER    I. 

The  General  Characteristics  of  Bacteria— Their  Morphology 

and  Structure — Vegetative  and  Spore  Forms     ...       33 

CHAPTER    II. 

The  Chemical  Composition  of  Bacteria — The  Conditions  Suit- 
able for  their  Growth       .......       50 

CHAPTER    III. 

Vital  Phenomena  of  Bacteria — Motion,  Heat,  and  Light  Pro- 
duction—Chemical Effects  ( Ferments,  Ptomai'ns,  Toxins)  .     58 

CHAPTER    IV. 

The  Relation  of  Bacteria  to  Disease        .....       85 

CHAPTER    V. 
Immunity       .         .         .         .         .         .         .         .         .         .     102 

CHAPTER    VI. 

Theories  of  Infection,  Immunity,  and  Recovery     .         .         .     118 

CHAPTER    VII. 
Infection  128 


viii  CONTENTS. 

CHAPTEK    VIII. 

PAGE 

The  Effect  of  Light,  Electricity,  Pressure,  Agitation,  Drying, 
and  Association  with  Other  Micro-organisms  upon  Bac- 
teria   134 

CHAPTEK    IX. 
Effect  of  Temperature  upon  Bacteria 144 

CHAPTER    X. 
The  Destruction  of  Bacteria  by  Chemicals      ....     151 

CHAPTEK    XL 

Practical  Disinfection  and  Sterilization  (House,  Person,  In- 
struments, and  Food) — Sterilization  of  Milk  for  Feeding 
Infants 168 

CHAPTEK    XII. 

The  Preparation,  Staining,  and  Microscopical   Examination 

of  Bacteria .     197 

CHAPTEK    XIII. 

The  Cultivation  of  Bacteria— Sterilization  of  Media — Appa- 
ratus    .  .212 

CHAPTEK    XIV. 

The  Use  of  Animals  for  Diagnostic  and  Test  Purposes  .         .     236 

CHAPTER    XV. 

The  Procuring  of  Material  for  Bacteriological  Examination 

from  those  Suffering  from  Disease    .....     240 

CHAPTER    XVI. 

Bacteriological  Examination  of  Water  and  Air — The  Con- 
tamination and  Purification  of  Drinking  Waters  .  .  245 


CONTENTS  ix 

CHAPTER    XVII. 

PAGE 

The  Classification  of  Bacteria          ......     257 

CHAPTER    XVIII. 

Bacillus  of  Tuberculosis  (Koch's  Tubercle  Bacillus)       .         .     263 

CHAPTER    XIX. 

Bacilli  Showing  Similar  Staining  Reactions  to  those  of  the 
Tubercle  Bacilli— Syphilis  Bacillus— Smegma  Bacillus — 
Leprosy  Bacillus— Grass  Bacilli 311 

CHAPTER    XX. 
Influenza  Bacillus  .........     320 

CHAPTER    XXI. 
Diphtheria  Bacillus 329 

CHAPTER    XXII. 
Tetanus  Bacillus 335 

CHAPTER    XXIII. 

Bacillus    Typhosus    ( Eberth-Gaff ky's    Bacillus   of    Typhoid 

Fever  ;  Bacillus  Typhi  Abdominalis)       ....     402 

CHAPTER    XXIV. 

Bacillus  Coli  Comnmnis  (or  Colon  Bacillus  of  Escherich)       .     444 

CHAPTER    XXV. 

Pneumobacillus  ( Friedlander's  Bacillus)          .         .  .     455 

CHAPTER    XXVI. 

The   Producers  of  Abscess,   Cellulitis,   Septicaemia,  etc.   (the 

Staphylococci  and  Micrococcus  Tetragenus)      .         .         .     461 


x  CONTENTS. 

CHAPTEE    XXVII. 

PAGE 

Streptococcus  Pyogenes  (Streptococcus  Erysipelatis  ;  Strep- 
tococcus of  Pus  ;  Streptococcus  Pathogenes  Longus)  .  476 

CHAPTER    XXVIII. 

Micrococcus  Lanceolatus  (Pneumococcus  ;  Micrococcus  Pneu- 
moniae  Crouposae  of  Sternberg ;  Micrococcus  of  Sputum 
Septicaemia  and  Diplococcus  of  Fraenkel ;  Diplococcus 
Pneumonias  of  Weichselbaum )  .  .  .  -  .  498 

CHAPTEE    XXIX. 

Diplococcus  Intracellularis  Meningitidis          .        .         .         .     516 

CHAPTER  XXX. 

Micrococcus  Gonorrhoaae  (Gonococcus  Neisser)       .         .         .     522 

CHAPTER    XXXI. 

Bacillus  Pyocyaneus  (Bacillus  of  Green  and  of  Blue  Pus) — 
Bacillus  Proteus  Vulgaris — Bacillus  of  Malignant  (Edema 
— Bacillus  Aerogenes  Capsulatus  .  ....  .  535 

CHAPTER    XXXII. 

Bacillus  Anthracis    (Anthrax   Bacillus) — Bacillus  Anthracis 

Symptomatici .         .         .     547 

CHAPTER    XXXIII. 

Spirillum    Choleras    Asiaticae    (Koch's    Comma    Bacillus   of 

Asiatic  Cholera) 568 

CHAPTER    XXXIV. 

Spirilla  Resembling  that  of  Cholera — The  Spirillum  of  Re- 
lapsing Fever 589 


CONTENTS.  xi 

CHAPTER    XXXV. 

PAGE 

Glanders  Bacillus 598 

CHAPTER    XXXVI. 

Bubonic  Plague  Bacillus — Yellow  Fever   Bacillus — Whoop- 
ing-cough Bacillus .         .     606 


APPENDIX. 

BRIEF  DESCRIPTIONS  OF   A   FEW  REPRESENTATIVE 

PATHOGENIC  MICRO-ORGANISMS  WHICH 

ARE  NOT  BACTERIA 

CHAPTER    XXXVII. 

The  Streptothrix  Group  ( Actinomyces) — Favus  and  Ring- 
worm Fungi  —Yeast  .......  615 

CHAPTER    XXXVIII. 

Plasmodium  Malarise  (Malarial  Parasites;  Laverania)  — 
Amoeba  Coli  (Amoeba  Dysenteriae  of  Councilman  and 
Lafleur  ;  Dysenteric  Amoeba) 626 

CHAPTER    XXXIX. 

Vaccine — The  Micro-organism  of  Smallpox  and  Cowpox         .     648 

CHAPTER     XL. 
Rabies  (Hydrophobia) 659 


BACTERIOLOGY  IN  MEDICINE  AND  SURGERY. 


INTRODUCTION. 

ALTHOUGH  most  of  the  more  important  discoveries 
in  bacteriology  which  place  it  on  the  footing  of  a 
science  are  of  comparatively  recent  date,  the  founda- 
tion of  the  study  of  vegetable  and  other  micro-organisms 
was  laid  over  two  centuries  ago.  From  the  earliest 
times  its  history  has  been  intimately  associated  with 
that  of  medicine.  Indeed,  it  is  only  through  the  inves- 
tigations into  the  life-history  of  micro-organisms  in  their 
relation  to  disease  that  our  present  knowledge  of  the 
etiology,  course,  and  prevention  of  the  infectious  diseases 
has  been  acquired  ;  and  it  is  only  by  the  practical  ap- 
plication of  the  principles  and  methods  of  bacteriology 
that  many  diseases  can  be  positively  diagnosed  or  the 
problems  which  present  themselves  to  the  sanitarian  be 
certainly  solved.  The  prominent  position  which  bac- 
teriology already  holds  toward  medicine  is,  moreover, 
daily  increasing  in  importance.  Original  discoveries  are 
constantly  adding  to  the  list  of  known  germ  diseases, 
and  the  outlook  is  favorable  for  eventually  obtaining 
through  serums  or  through  the  toxic  substances  of  the 
micro-organisms  themselves  means  for  immunizing 
against,  if  not  curing,  many  of  the  specific  infections. 
Even  at  present  bacterial  products  and  protective 

2 


18  BACTERIOLOGY. 

serums  are  used  successfully  as  preventives  in  many  of 
the  infectious  diseases  and  as  a  cure  in  several.  An 
acquaintance,  therefore,  with  the  main  facts  and  results 
of  bacteriology  is  as  necessary  to  the  education  of  the 
modern  physician  as  a  knowledge  of  anatomy,  pathol- 
ogy, chemistry,  or  any  of  the  allied  sciences. 

But  before  entering  into  a  detailed  consideration  of 
the  subject,  it  may  be  interesting  and  instructive  to 
review  briefly  the  most  important  steps  which  led  up 
to  the  development  of  the  science,  and  upon  which  its 
foundation  rests,  in  which  we  shall  see  that  the  vast 
results  obtained  by  bacteriology  were  gained  only 
through  long  and  laborious  research,  and  after  many 
obstacles  were  met  and  overcome  by  indomitable  per- 
severance and  accurate  observation  and  experiment. 

The  first  authentic  observations  of  living  microscopi- 
cal organisms  of  which  there  is  any  record  are  those  of 
Athanasius  Kircher,  in  1671.  This  original  investi- 
gator demonstrated  the  presence  in  putrid  meat,  milk, 
vinegar,  cheese,  etc.,  of  <e  minute  living  worms,"  but 
did  not  describe  their  form  or  character. 

Not  long  after  this,  in  1675,  Anthony  von  Leeuwen- 
hoek  observed  in  rain-water  putrid  infusions,  and  in 
his  own  and  other  saliva  and  diarrhoeal  evacuations 
living,  motile  "  animalculse  "  of  most  minute  dimen- 
sions, which  he  described  and  illustrated  by  drawings. 
Leeuwenhoek  was  a  linen-draper  by  trade,  living  at 
the  time  of  his  discoveries  in  Amsterdam,  but  he  prac- 
tised the  art  of  lens-grinding,  in  which  he  eventually 
became  so  proficient  that  he  perfected  a  lens  superior 
to  any  magnifying  glass  obtainable  at  that  day,  and 
with  which  he  was  enabled  to  see  objects  very  much 
smaller  than  had  ever  been  seen  before.  "  With  the 


INTRODUCTION.  19 

greatest  astonishment/'  he  writes,  "  I  observed  distrib- 
uted everywhere  through  the  material  which  I  was 
examining  animalcules  of  the  most  microscopic  size, 
which  moved  themselves  about  very  energetically." 
The  work  of  this  observer  is  conspicuous  for  its  purely 
objective  character  and  absence  of  speculation  ;  and  his 
descriptions  and  illustrations  are  done  with  remarka- 
ble clearness  and  accuracy,  considering  the  imperfect 
optical  instruments  at  his  command.  There  is  little 
doubt  that  Leeuwenhoek  really  saw  some  of  the  larger 
species  of  micro-organisms  which  we  now  recognize  as 
bacteria,  probably  spirilla. 

It  was  not  until  many  years  later,  however,  that  any 
attempt  was  made  to  define  the  characters  of  these 
minute  organisms  and  to  classify  them.  The  first  to 
make  such  an  effort  was  Otto  Friedrich  Miiller,  in 
1786  ;  but  having  no  means  of  obtaining  pure  cultures 
all  the  earlier  botanists  naturally  fell  into  serious  errors 
in  the  classification  of  bacteria.  Thus  various  motile 
organisms,  which  are  now  known  to  be  of  vegetable 
origin,  were  commonly  included  under  the  infusoria, 
which  are  unicellular  animal  organisms. 

Ehrenberg,  in  1838,  thus  describes  under  the  gen- 
eral name  Vibrioniens  four  genera  of  filamentous  bac- 
teria : 

1.  Bacterium — filaments  linear  and  inflexible. 

2.  Vibrio — filaments  linear,  sinuous,  flexible. 

3.  Spirillum — filaments  spiral,  inflexible.  y. 

4.  Sperochcete — filaments  spiral,  flexible. 
Dujardin,   in  1841,   also  placed  the  vibrioniens  of 

Ehrenberg  among  the  infusoria,  describing  them  as 
extremely  slender,  filiform  animals  without  appreciable 
organization  and  without  visible  locomotive  organs. 


20  BACTERIOLOGY. 

Perty,  in  1852,  drew  attention  to  the  vegetable 
origin  of  these  minute  organisms  ;  Robin,  in  1853, 
suggested  their  relationship  to  the  algae  ;  Davaine,  in 
1859,  emphasized  this  fact  ;  and  since  it  has  been  con- 
firmed by  the  investigations  of  Cohn,  Nageli,  and 
others.  Bacteria  are  now  generally  believed  by  bacte- 
riologists to  be  vegetable  organisms,  schizomycetes,  or 
fission-fungi,  closely  allied  to  the  algse. 

From  the  earliest  investigations  into  the  life-history 
and  properties  of  bacteria  these  micro-organisms  have 
been  thought  to  play  an  important  part  in  the  causa- 
tion of  infectious  diseases.  The  doctrine  of  contagium 
aminatum  was  based  upon  the  discoveries  of  Athana- 
sius  Kircherand  Leeuwenhoek,  and  the  "  animalculse  " 
then  observed  in  organic  materials  were  believed  to  be 
the  cause  of  the  great  epidemics  of  the  day,  such  as 
the  plague.  Shortly  after  these  first  investigations, 
Lange  and  Hauptmann  advanced  the  opinion  that 
puerperal  fever,  measles,  smallpox,  typhus,  pleurisy, 
epilepsy,  gout  and  many  other  diseases  were  due  to 
animal  contagion.  Andry  and  Linne,  in  1701,  as- 
sumed the  same  cause  for  syphilis,  and  Lancisi,  in 
1718,  for  malaria.  In  fact,  so  wide- spread  became  the 
belief  in  a  causal  relation  of  these  minute  organisms  to 
disease  that  it  soon  amounted  to  a  veritable  craze,  and 
all  forms  and  kinds  of  diseases  were  said  to  be  pro- 
duced in  this  way,  upon  no  other  foundation  than  that 
these  organisms  had  been  found  in  the  mouth  and  in- 
testinal contents  of  men  and  animals,  and  in  water. 

Among  those  who  were  especially  conspicuous  at 
this  time  for  their  advanced  views  on  the  germ-theory 
of  infectious  diseases  was  Marcus  Antonius  Plenciz,  a 
physician  of  Vienna.  This  acute  observer,  who  pub- 


INTRODUCTION.  21 

lished  his  views  in  1762,  maintained  that  not  only 
were  all  infectious  diseases  caused  by  micro-organisms, 
but  that  the  infective  material  could  be  nothing  else 
than  a  living  organism.  On  these  grounds  he  en- 
deavored to  explain  the  variations  in  the  period  of 
incubation  of  the  different  infectious  diseases.  He  also 
insisted  that  there  were  special  germs  for  each  infectious 
disease  by  which  the  specific  disease  was  produced. 
Plenciz  believed,  moreover,  that  these  organisms  were 
capable  of  multiplication  in  the  body,  and  suggested 
the  possibility  of  their  being  conveyed  from  place  to 
place  through  the  air.  He  also  made  original  investi- 
gations into  the  process  of  decomposition,  and  having 
found  "  animalcube  "  in  all  decomposing  matter,  he 
became  so  thoroughly  convinced  of  the  causative  rela- 
tion of  these  organisms  to  the  process  that  he  formu- 
lated the  law  that  decomposition  takes  place  by  means 
of  living  organisms,  and  is  possible  only  through  their 
increase. 

These  views,  it  is  true,  were  largely  speculative,  and 
rested  upon  insufficient  experiment;  but  they  were  so 
plausible,  and  the  arguments  put  forward  in  their  sup- 
port were  so  logical  and  convincing,  that  they  continued 
to  gain  ground,  in  spite  of  considerable  opposition  and 
ridicule,  and  in  many  instances  the  conclusions  reached 
have  since  been  proved  to  be  correct.  The  fact  that 
infectious  diseases  were  of  sudden  occurrence,  breaking 
out  often  in  isolated  places,  and  that  they  frequently 
remained  clinging  for  long  periods  to  certain  localities, 
leaving  others  unaffected,  was  evidence  that  they  were 
not  produced  by  a  gaseous  infective  agent.  Moreover, 
the  mode  of  infection,  its  unlimited  development  among 
large  numbers  of  individuals,  and  gradual  spread  over 


22  BACTERIOLOGY. 

wide  areas — the  incubation,  course  of,  and  resulting 
immunity  in  recovery  from  infectious  diseases — all 
pointed  to  the  probable  cause  being  a  living  organism. 

Amoug  other  distinguished  men  of  the  day  whose 
observations  exerted  a  most  powerful  influence  upon 
the  doctrine  of  infection  may  be  mentioned  Henle. 
His  writings  (Pathological  Investigations,  1840,  and 
Text-book  of  Rational  Pathology,  1853),  in  which  he 
described  the  relation  of  micro-organisms  to  infectious 
diseases,  and  defined  the  character  and  action  of  bac- 
teria upon  certain  phases  and  symptoms  of  these  affec- 
tions, are  remarkable  for  their  clearness  and  precision. 

But,  meanwhile,  the  question  which  most  interested 
these  investigators  into  the  cause  of  infectious  diseases 
was,  Whence  are  these  micro-organisms  derived  which 
were  supposed  to  produce  them  ?  Were  they  the  result 
of  spontaneous  generation  due  to  vegetative  changes  in 
the  substances  in  which  the  organisms  were  found,  or 
were  they  reproduced  from  similar  pre-existing  organ- 
isms—the so-called  vitalistic  theory  ?  This  question  is 
intimately  connected  with  the  investigations  into  the 
origin  and  nature  of  fermentation  and  putrefaction. 

Among  those  who  advocated  the  theory  of  spon- 
taneous generation  was  Neidham,  who,  in  1749,  at- 
tempted to  prove  by  experiment  the  truth  of  his  opin- 
ions. He  placed  a  grain  of  barley  in  a  watch-glass 
containing  water,  covered  it  carefully,  and  allowed  it 
to  germinate.  On  later  examination  he  found  bacteria 
present,  which  he  maintained  were  the  result  of  changes 
in  the  grain  itself  due  to  its  germination. 

In  1769,  Spallanzani  showed  by  another  experiment 
that  the  theory  of  spontaneous  generation  was  incor- 
rect. He  demonstrated  that  if  putrescible  infusions 


INTR  OD  UCTION.  23 

of  organic  matter  were  placed  in  symmetrically  sealed 
flasks  and  then  boiled  the  liquids  were  sterilized  ; 
neither  were  living  organisms  found  in  the  solutions, 
nor  did  they  decompose  ;  and  the  infusions  remained 
unchanged  for  an  indefinite  period. 

It  was  objected  to  these  experiments  that  the  high 
temperature  to  which  the  liquids  had  been  subjected  so 
altered  them  that  spontaneous  generation  could  no 
longer  take  place.  This  objection  was  met  by  Spall- 
anzani  by  cracking  one  of  the  flasks  and  allowing  air 
to  enter,  when  living  organisms  and  decomposition 
again  appeared  in  the  boiled  infusions. 

Another  objection  raised  was  that  in  excluding  the 
oxygen  of  the  air  by  hermetically  sealing  the  flasks  the 
essential  condition  for  the  development  of  fermentation, 
which  required  free  admission  of  this  gas,  was  inter- 
fered with.  This  objection  was  then  met  by  Schulze, 
in  1836,  by  causing  the  air  admitted  to  the  boiled 
decomposable  liquids  to  pass  through  strong  sulphuric 
acid.  Air  thus  robbed  of  its  living  organisms  did  not 
produce  decomposition  ;  whereas  when  no  such  precau- 
tions were  taken  with  the  air  admitted  the  boiled  solu- 
tions quickly  fell  into  putrefaction,  and  living  organ- 
isms were  found  to  be  present. 

Schwann,  in  1839,  obtained  similar  results  in  another 
way;  he  deprived  the  air  admitted  to  his  boiled  liquids 
of  micro-organisms  by  passing  it  through  a  tube 
which  was  heated  to  a  temperature  high  enough  to 
destroy  them.  To  this  investigator  is  also  due  the 
credit  of  having  discovered  the  specific  cause — the  yeast 
plant,  or  saccharomyces  cerevisice — of  alcoholic  fermen- 
tation, the  process  by  which  sugar  is  decomposed  into 
alcohol  and  carbonic  acid. 


24  BACTERIOLOGY. 

Helmholtz,  in  1843,  repeated  and  confirmed 
Schwann's  experiments  with  calcined  air.  He  found 
that  the  free  admission  of  air  so  treated  to  boiled 
organic  infusions  was  not  capable  of  producing  fer- 
mentation of  any  kind. 

Again,  it  was  objected  to  these  experiments  that  the 
heating  of  the  air  had  perhaps  brought  about  some 
chemical  change  which  hindered  the  production  of  fer- 
mentation. Schroeder  and  von  Dusch,  in  1854,  then 
showed  that  by  a  simple  process  of  filtration,  which 
has  since  proved  of  inestimable  value  in  bacteriological 
work,  the  air  can  be  mechanically  freed  from  germs. 
By  placing  in  the  mouth  of  the  flask  containing  the 
boiled  solutions  a  loose  plug  of  cotton,  through  which 
the  air  could  freely  circulate,  it  was  found  that  all 
suspended  micro-organisms  could  be  excluded,  and 
that  air  passed  through  such  a  filter,  whether  hot  or 
cold,  did  not  cause  fermentation  of  boiled  infusions. 

Similar  results  were  obtained  by  Hoffmann  in  1860, 
and  by  Chevreul  and  Pasteur  in  1861,  without  a  cotton 
filter,  by  drawing  out  the  neck  of  the  flask  to  a  fine 
tube  and  turning  it  downward,  leaving  the  mouth  open. 
In  this  case  the  force  of  gravity  prevents  the  suspended 
bacteria  from  ascending,  and  there  is  no  current  of  air 
to  carry  them  upward  through  the  tube  into  the  flask 
containing  the  boiled  infusion. 

Tyndall  later  showed  (1876),  by  his  well-known  in- 
vestigations upon  the  floating  matters  of  the  air,  that 
in  a  closed  chamber,  in  which  the  air  is  not  disturbed 
by  currents,  all  suspended  particles  settle  to  the  bottom, 
the  superincumbent  air  being  optically  pure,  as  is 
proved  by  passing  a  ray  of  light  through  it.  He  dem- 
onstrated that  the  presence  of  living  organisms  in 


INTR  OD  UCTION.  25 

decomposing  fluids  was  always  to  be  explained  either 
by  the  pre-existence  of  similar  living  forms  in  the  in- 
fusion or  upon  the  walls  of  the  vessel  containing  it,  or 
by  the  infusion  having  been  exposed  to  air  which  was 
contaminated  with  organisms. 

These  facts  have  since  been  practically  confirmed  on 
an  enormous  scale  in  the  preservation  of  food  by  the 
process  of  sterilization.  Indeed,  there  is  scarcely  any 
biological  problem  which  has  been  so  satisfactorily 
solved  or  in  which  such  uniform  results  have  been  ob- 
tained ;  but  all  through  the  experiments  of  the  earlier 
investigators  irregularities  were  constantly  appearing. 
Although  in  the  large  majority  of  cases  it  was  found 
possible  to  keep  boiled  organic  liquids  sterile  in  flasks 
to  which  the  oxygen  of  the  air  had  free  access,  the 
question  of  spontaneous  generation  still  remained  un- 
settled, inasmuch  as  occasionally,  even  under  the  most 
careful  precautions,  decomposition  did  occur  in  such 
boiled  liquids. 

This  fact  was  explained  by  Pasteur  in  I860  by  ex- 
periments showing  that  the  temperature  of  boiling 
water  was  not  sufficient  to  destroy  all  living  organ- 
isms, and  that,  especially  in  alkaline  liquids,  a  higher 
temperature  was  required  to  insure  sterilization.  He 
showed  that  at  a  temperature  of  110°  to  112°  C.  (230° 
to  233°  F.),  however,  which  he  obtained  by  boiling 
under  a  pressure  of  one  and  one-half  atmospheres,  all 
living  organisms  were  invariably  killed. 

Pasteur  at  a  later  date  (1865)  demonstrated  that  the 
organisms  which  resist  the  boiling  temperature  are,  in 
fact,  reproductive  bodies,  which  he  described  under  the 
name  of  "  corpuscles  ovoides  "  or  "  corpuscles  bril- 
lants  " — now  known  as  spores.  Perty,  in  1852,  and 


26  BACTERIOLOGY. 

Robin,  in  1853,  had  observed  these  highly  refractile 
bodies  ;  but  it  was  not  until  1876  that  the  development 
of  spores  was  carefully  investigated  and  explained  by 
Cohn  and  later  by  Koch.  These  observers  showed  that 
certain  rod-shaped  organisms  possess  the  power  of  pass- 
ing into  a  resting  or  spore-stage  under  peculiar  conditions 
of  growth,  and  when  in  this  stage  they  are  much  less 
susceptible  to  the  injurious  action  of  higher  tempera- 
tures than  when  in  their  normal  vegelative  condition. 

With  this  discovery  the  controversy  of  spontaneous 
generation  was  finally  settled.  If  these  micro-organ- 
isms, some  of  them  being  capable  of  producing  the 
more  resistant  spores,  were  present  in  the  air,  dust, 
soil,  water,  etc.,  it  was  easy  enough  to  explain  the 
irregularities  in  the  foregoing  experiments  ;  nor  was  it 
any  longer  to  be  doubted  that  these  bacteria,  through 
their  products,  were  the  cause,  not  the  effect,  of  fer- 
mentation and  putrefaction,  and  that  when  organic  sub- 
stances were  completely  sterlized  and  protected  against 
the  entrance  of  living  germs  from  without,  no  develop- 
ment of  micro-organisms  occurred  in  them. 

Stimulated  by  the  establishment  of  the  fact  that  fer- 
mentation and  putrefaction  were  due  to  the  action  of 
living  organisms  reproduced  from  similar  pre-existing 
forms,  the  study  of  the  causal  relation  of  these  micro- 
organisms to  disease  was  taken  up  with  renewed  vigor. 
Reference  has  already  been  made  to  the  opinions  and 
hypothesis  of  the  earlier  observers  as  to  the  microbic 
origin  of  infectious  diseases.  The  first  positive  grounds, 
however,  for  this  doctrine,  founded  upon  actual  ex- 
periment, were  the  investigations  into  the  cause  of  cer- 
tain infectious  diseases  in  insects  and  plants.  Thus 
Bassi,  in  1837,  demonstrated  that  a  fatal  infectious 


INTRODUCTION.  27 

malady  of  the  silkworm — muscardine — was  due  to  a 
parasitic  micro-organism.  Pasteur  later  devoted  several 
years'  study  to  an  exhaustive  investigation  into  the 
same  subject  ;  and  in  like  manner  Tulasse,  in  1864, 
and  Kiihne,  in  1855,  showed  that  certain  specific  affec- 
tions in  grains,  the  potato,  etc.,  were  due  to  the  inva- 
sion of  parasites. 

Very  soon  after  this  it  was  demonstrated  that  micro- 
organisms were  the  cause  of  certain  infectious  diseases 
in  man  and  the  higher  animals.  Bacteriological  re- 
search has  always  been  of  special  interest  to  physicians. 
Many  of  the  most  distinguished  physicians  of  the  day, 
in  the  earlier  history  of  the  science,  concerned  them- 
selves in  these  investigations,  and  the  progress  made 
during  the  past  fifteen  or  twenty  years  has  been  largely 
due  to  their  work.  Davaine,  a  famous  French  physi- 
cian, has  the  honor  of  having  first  demonstrated  the 
causal  relation  of  a  micro-organism  to  a  specific  infec- 
tious disease  in  man  and  animals.  The  anthrax  bacil- 
lus was  discovered  in  the  blood  of  animals  dying  from 
this  disease  by  Pollender,  in  1849,  and  by  Davaine, 
in  1850 ;  but  it  was  not  until  1863  that  the  last-named 
observer  demonstrated  by  inoculation  experiments  that 
the  bacillus  was  the  cause  of  anthrax.  These  experi- 
ments were  subsequently  confirmed  by  Pasteur,  Koch, 
and  others. 

The  next  discoveries  made  were  those  relating  to 
wounds  and  the  infections  to  which  they  are  liable. 
Rindfleisch,  in  1866,  and  Waldeyer  and  von  Reckling- 
hausen,  in  1871,  were  the  first  to  draw  attention  to  the 
minute  organisms  occurring  in  the  pysemic  processes 
resulting  from  infected  wounds,  and  occasionally  fol- 
lowing typhoid  fever.  Further  operations  were  made 


28  BACTERIOLOGY. 

in  erysipelatous  inflammations  secondary  to  injury  by 
Wilde,  Orth,  von  Recklinghausen,  Luthomsky,  Bill- 
roth,  Ehrlich,  Fehleisen,  and  others,  agreeing  that  in 
these  conditions  micro-organisms  could  always  be  de- 
tected in  the  lymph-channels  of  the  subcutaneous 
tissues  ;  and  through  numerous  experiments  on  ani- 
mals the  pathogenic  character  of  the  micro-organisms 
found  in  erysipelas,  suppuration  from  wounds,  diph- 
theria, puerperal  fever,  etc.,  was  established  by  Oertel, 
Huester,  Birsch-Hirschfeld,  Narsiloff,  Classen,  Letz- 
erich,  Leber,  Frisch,  Eberth,  Klebs  and  others. 

The  brilliant  results  obtained  by  Lister,  in  1863— 
1870,  in  the  antiseptic  treatment  of  wounds,  to  prevent 
or  inhibit  the  action  of  infective  organisms,  exerted  a 
powerful  influence  on  the  doctrine  of  bacterial  infec- 
tion, causing  it  to  be  recognized  far  and  wide  and 
gradually  lessening  the  number  of  its  opponents. 

The  next  important  discovery  was  that  of  Ober- 
meier,  a  German  physician,  who,  in  1873,  announced 
having  found  in  the  blood  of  patients  suffering  from 
relapsing  fever  a  minute  spiral,  actively  motile  micro- 
organism— the  spirochcete  Obermeieri — which  is  now 
generally  recognized  as  the  specific  infectious  agent  in 
this  disease. 

In  1877,  Weigert  and  Ehrlich  recommended  the  use 
of  the  aniline  dyes  as  staining  agents  in  the  micro- 
scopical examination  of  micro-organisms  in  cover-glass 
preparations. 

In  1878,  Koch  published  his  important  work  on 
traumatic  infectious  diseases. 

Hausen,  in  1879,  reported  the  discovery  of  bacilli 
in  the  cells  of  leprous  tubercles,  which,  from  subsequent 
researches,  are  believed  to  be  the  cause  of  leprosy. 


INTRODUCTION.  29 

Neisser,  in  the  same  year  (1879),  discovered  the 
"  gonococcus  "  in  gonorrhoeal  discharges. 

In  1880,  Eberth  and  Koch  independently  observed 
the  typhoid  bacillus,  but  it  was  not  until  1884  that 
Gaffky  published  his  important  researches,  and  proved 
the  etiological  relation  of  this  bacillus  to  typhoid  fever. 

In  the  same  year  (1880)  several  important  communi- 
cations in  bacteriological  research  appeared.  Pasteur 
published  his  discovery  of  the  bacillus  of  fowl  cholera 
and  his  investigations  upon  the  attenuation  of  the  virus 
of  anthrax  and  of  fowl  cholera,  and  upon  protective 
inoculation  against  these  diseases. 

Sternberg  and  Pasteur  independently  observed  (1880) 
a  pathogenic  micrococcus  in  saliva,  which  was  subse- 
quently proved  by  Fraenkel  and  others  (1885)  to  be 
the  organism  most  commonly  associated  with  acute 
croupous  pneumonia — the  "  diplococcus  pneumoniae" — 
and  now  recognized  as  the  usual  cause  of  that  disease. 

In  1881,  Koch  made  his  fundamental  researches  upon 
pathogenic  bacteria,  the  result  of  which  was  the  estab- 
lishment of  a  foundation  upon  which  bacteriology  of  the 
future  was  to  rest.  He  introduced  solid  culture  media 
and  the  "  plate  method7'  for  obtaining  pure  cultures, 
and  showed  how  different  organisms  could  be  isolated, 
cultivated  independently,  and  by  inoculation  of  pure 
cultures  into  susceptible  animals  made,  in  many  cases, 
to  reproduce  the  specific  disease  of  which  they  were  the 
cause  ;  and  he  laid  down  the  laws  by  which  it  may  be 
proved  that  a  micro-organism  is  the  specific  cause  of 
a  disease.  It  was  in  the  course  of  this  work  that  the 
Abbe  system  of  substage  condensing  apparatus  was  first 
used  in  bacteriology  and  that  Weigert's  method  of 
staining  was  generally  employed. 


30  BACTEEIOLOG  T. 

In  1882,  Koch  published  his  discovery  of  the  tubercle 
bacillus. 

The  same  year  (1882)  Pasteur  published  his  in- 
vestigations upon  "rouget"  or  hog  erysipelas.  In 
this  year,  also,  his  first  communication  upon  rabies 
appeared. 

In  1882,  also,  Loemer  and  Schiitz  discovered  the 
bacillus  of  glanders. 

The  cholera  spirillum,  or  "  comma  bacillus/'  was 
discovered  by  Koch  in  1884. 

The  diphtheria  bacillus  was  discovered  during  the 
same  year  (1884)  by  Loemer,  though  it  had  been  ob- 
served by  Klebs  the  year  before  (1883). 

Rosenbach,  in  1884,  by  the  application  of  Koch's 
methods,  fixed  definitely  the  characters  of  the  various 
micro-organisms  found  in  the  pus  from  acute  abscesses, 
etc. 

The  tetanus  bacillus  was  also  discovered  in  1884  by 
Nicolaier.  Carle  and  Rattone  showed  that  tetanus  is 
an  infectious  disease  communicable  to  man  by  inocula- 
tion. Kitasato,  in  1889,  obtained  the  bacillus  in  pure 
cultures. 

In  1892,  Pfeiffer  and  Canon  independently  discov- 
ered a  bacillus  which  is  believed  to  be  the  specific 
cause  of  influenza. 

In  1894,  Kitasato,  the  Japanese  bacteriologist,  during 
a  visit  to  China,  discovered  the  bacillus  of  the  bubonic 
plague. 

These  include  all  the  most  important  pathogenic 
bacteria,  the  discovery  of  which  is  of  special  interest 
to  medical  students  and  physicians.  We  cannot  close 
this  brief  historical  review,  however,  of  the  progress  of 
our  knowledge  in  this  department  of  science,  without 


INTR  OD  UCTION.  31 

referring  to  the  recent  discovery  of  the  antitoxins  of 
diphtheria  and  tetanus,  the  protective  inoculations 
against  rabies,  the  plague,  cholera,  etc.,  and  the  pecu- 
liar characteristics  of  the  serum  of  those  ill  with  infec- 
tious diseases.  These  discoveries,  in  which  the  names 
of  Pasteur,  Koch,  Behring,  Kitasato,  Roux,  Pfeiffer, 
and  Widal  are  among  the  most  prominent,  mark  an 
epoch  in  the  history  of  bacteriology  and  scientific  medi- 
cine. Lately,  attention  has  also  been  given  to  the 
smaller  group  of  the  animal  parasites,  the  protozoa, 
which  may  prove  to  be  the  source  of  infection  in  many 
diseases,  such  as  the  exanthemata,  in  one  of  which — 
smallpox — they  have  already  been  apparently  found. 


CHAPTER   I. 

THE      GENERAL      CHARACTERISTICS      OF      BACTERIA — 
THEIR  MORPHOLOGY  AND  CHEMICAL  COMPOSITION. 

BACTERIA  are  among  the  smallest  of  all  known  liv- 
ing organisms,  the  largest  of  them  having  a  diameter 
of  only  a  few  micromillimetres,  while  the  smallest  do 
not  measure  more  than  a  fraction  of  a  micromillimetre. 
Structurally  and  morphologically  they  are  extremely 
simple,  though  biologically  very  variable.  Through 
their  ability  to  derive  their  carbon  from  tartrates  and 
their  nitrogen  from  ammonia  or  its  salts,  they  are 
ranked  in  the  vegetable  kingdom.  They  obtain  their 
food  entirely  through  the  surface  absorption  of  soluble 
nutritious  substances.  They  are  reproduced  by  trans- 
verse division,  and  in  some  respects  resemble  the  fungi  ; 
hence  called  by  Nageli  fission-fungi,  or  schizomycetes. 
They  are  also  closely  allied  to  certain  kinds  of  algae, 
though  they  must  receive  their  nourishment  from  living 
or  dead  organic  material,  since  they  are  without  chloro- 
phyll, the  green  coloring  matter  possessed  by  the  higher 
plants,  by  means  of  which  they  are  enabled,  in  the 
presence  of  sunlight,  to  decompose  CO2,  NH3,  and  H2S 
into  their  elementary  constituents.  A  few  varieties  of 
unicellular  organisms  resemble  bacteria  in  all  their 
known  characteristics,  except  that  they  possess  chloro- 
phyll or  substances  similar  to  it.  Others,  still,  which 
have  no  chlorophyll,  are  able  in  the  absence  of  light  to 
build  up  organic  substances  synthetically.  Bacteria, 

3 


34  BACTERIOLOGY. 

especially  the  motile  forms,  are  also  closely  allied  to 
some  of  the  micro-organisms  which  belong  to  the  ani- 
mal kingdom.  If  we  exclude  the  micro-organisms 
containing  chlorophyll,  bacteria  may  be  defined  as 
extremely  minute  vegetable  organisms;  without  chloro- 
phyll, consisting  of  single  spherical,  rod-shaped,  or 
corkscrew-like  cells  or  aggregates  of  such  cells,  between 
whose  protoplasm  and  nucleus  it  has  been  as  yet  im- 
possible to  differentiate  with  certainty. 

Bacteria  occur  as  saprophytes  or  refuse-eaters  and  as 
parasites.  Saprophytic  bacteria  are  such  as  commonly 
exist  independently  of  a  living  host,  obtaining  their 
supply  of  nutriment  from  soluble  food-stuffs  in  dead 
organic  matter.  Parasitic  bacteria,  on  the  other  hand, 
live  on  or  in  some  other  organism,  from  which  they 
derive  their  nourishment  for  the  whole  or  a  part  of 
their  existence.  Those  bacteria  which  depend  entirely 
upon  a  living  host  for  their  existence  are  known  as 
strict  parasites  ;  those  which  can  lead  a  saprophytic 
existence,  but  which  can  also  thrive  within  the  body 
of  a  living  animal,  are  called  facultative  parasites. 
The  strict  saprophytes,  which  represent  the  large 
majority  of  all  bacteria,  while  they  destroy  refuse,  are 
not  only  harmless  to  living  organisms  but  perform 
many  important  functions  in  nature  without  which 
existence  would  be  impossible,  such  as  the  destruction 
of  dead  organic  material  through  decomposition,  putre- 
faction, and  fermentation.  The  parasites,  on  the  con- 
trary, though  some  of  them  may  multiply  in  the  secre- 
tions or  on  the  surface  of  the  body  without  injury  to 
the  animal  upon  which  they  depend  for  their  exist- 
ence, are  usually  harmful  invaders,  giving  rise  through 
tHe  lesions  brought  about  in  the  body  tissues  by  their 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      35 

growth  and  products  to  derangements  which  are  known 
as  acute  or  chronic  infectious  diseases. 

Numerous  attempts  have  been  made  by  various 
authors  to  classify  bacteria  systematically,  but  usually 
with  the  proviso  that  the  system  was  only  a  temporary 
one.  The  classification  of  the  older  naturalists  and 
botanists  was  based  generally  upon  purely  morphological 
peculiarities.  As  this  depended,  at  times,  upon  slight 
variations  that  were  seen  to  occur  in  the  size  and  shape 
of  one  and  the  same  species,  it  naturally  resulted  in  a 
more  or  less  complicated  arrangement.  In  this  place 
the  morphological  character  of  the  bacteria  will  alone 
be  given,  their  classification  being  left  until  the  general 
characteristics  of  bacteria  have  been  considered. 

ih 
MORPHOLOGY. 

The  basic  forms  of  the  single  bacterial  cells  are 
threefold — the  sphere,  the  rod,  and  the  segment  of  a 
spiral.  Although  under  different  conditions,  the  form 
of  any  one  species  may  vary  considerably,  yet  these 
three  main  divisions  under  similar  conditions  are  per- 
manent; and,  so  far  as  we  know,  it  is  never  possible 
by  any  means  to  bring  about  changes  in  the  organisms 
that  will  result  in  the  conversion  of  the  morphology  of 
the  members  of  one  group  into  that  of  another — that 
is,  micrococci  always,  under  suitable  conditions,  produce 
micrococci,  bacilli  produce  bacilli,  and  spirilla  produce 
spirilla. 

The  form  of  the  bacterial  cells  at  their  stage  of  com- 
plete development  must  be  distinguished  from  that 
which  they  possess  just  after  or  just  before  they  have 
divided.  As  the  spherical  cell  develops  preparatory  to 


36  BACTERIOLOGY. 

its  division  into  two  cells  it  becomes  elongated  and  ap- 
pears as  a  short  oval  rod;  at  the  moment  of  its  division, 
on  the  contrary,  the  transverse  diameter  of  each  of  its 
two  halves  is  greater  than  their  long  diameter.  A 
short  rod  becomes  in  the  same  way,  at  the  moment 
of  its  division,  two  cells,  the  long  diameter  of  each  of 
which  may  be  even  a  trifle  less  than  its  short  diameter, 
and  thus  they  appear  on  superficial  examination  as 
spheres. 

As  bacteria  multiply  the  cells  produced  from  the 
parent  cell  have  a  greater  or  less  tendency  to  remain 
attached.  In  some  varieties  this  tendency  is  extremely 
slight,  in  others  it  is  marked.  This  union  may  appear 
simply  as  an  aggregation  of  separate  bacteria  or  so  close 
that  the  group  appears  as  a  single  cell.  According  to 
the  method  of  the  cell  division  and  the  tenacity  with 
which  the  cells  hold  together,  we  get  different  group- 
ings of  bacteria,  which  aid  us  in  their  differentiation  and 
identification.  Thus  whether  the  bacterial  cell  divides 
in  one,  two,  or  three  planes,  we  get  forms  built  in 
one,  two,  or  three  dimensions.  If  we  group  bacteria 
according  to  the  characteristic  form  of  the  cells,  and 
then  subdivide  them  according  to  the  manner  of  their 
division  in  reproduction  and  the  tenacity  with  which 
the  newly  developed  cells  cling  to  one  another,  we  will 
have  the  following  varieties  : 

1.  Spherical  Form,  or  Coccus  (Figs.  1  to  4).  The  size 
varies  from  about  Q.3/J.  as  minimum  diameter  to  3//  as 
maximum.  The  single  elements  are  at  the  moment  of 
their  complete  development,  so  far  as  we  can  deter- 
mine, absolutely  spherical;  but  when  seen  in  the  process 
of  multiplication  through  division  the  form  is  seldom 
that  of  a  true  sphere.  Here  we  have  elongated  or  lancet- 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      37 

shaped  forms,  as  frequently  seen  in  the  diplococcus  of 
pneumonia,  or  the  opposite,  as  in  the  diplococcus  of 
gonorrhoea,  where  the  cocci  appear  to  be  flattened 
against  one  another.  Those  cells  which  divide  in  one 


FIG.  l, 


FIG.  2. 


****>     *"*«V. 


Single  coccus,  grouped  irregularly.       Diplococcus  of  pneumonia,  with  sur- 
Staphylococcus.  rounding  capsule. 


FIG.  3. 


FIG.  4. 


TBb 

J^m 

&$&L 


Streptococcus. 


Tetracoccus. 


direction  only  and  remain  attached  are  found  in  pairs 
(diplococci)  or  in  shorter  or  longer  chains  (streptococci). 
Those  which  divide  in  two  directions,  the  one  at  right 
angles  to  the  other,  form  bunches  of  four  (tetrads). 


38  BACTERIOLOGY. 

Those  which  divide  in  three  directions  and  cling  to- 
gether form  packets  in  cubes  (sarcinse).  Those  which 
apparently  divide  irregularly  in  any  axis  form  irregu- 
larly shaped,  grape-like  bunches  (staphylococci). 

There  are  a  considerable  number  of  bacteria  which 
appear  to  frequently  assume  spherical  forms,  or  at  least 
forms  so  like  spheres  that  they  cannot  be  differentiated 
from  them,  and  yet  under  other  conditions  they  generate 
rod-like  forms.  These  apparently  spherical  bacteria 
we  can  properly  regard  as  short  forms  of  bacilli,  which, 
owing  to  the  rapidity  of  division,  are  for  the  time  being 
of  the  same  size  in  both  diameters.  Under  suitable 
conditions,  however,  the  true  rod- shape  is  always  de- 
veloped. 

2.  Rod  Form,  or  Bacillus.  The  type  of  this  group  is 
the  cylinder.  The  length  of  the  fully  developed  cell 
is  always  longer  than  its  breadth.  The  size  of  the 
cells  of  different  varieties  varies  enormously,  from  a 
length  of  30//  and  a  breadth  of  4//  to  a  length  of  0.2// 
and  a  breadth  of  0.1  //.  The  largest  bacilli  met  with 
in  disease  do  not,  however,  average  over  3/*.  In  des- 
cribing their  forms  bacilli  are  roughly  classed  as  slender 
when  the  ratio  of  the  long  to  the  transverse  diameter  is 
from  1  :  4  to  1  :  10,  and  as  thick  when  the  proportions 
of  the  long  to  the  short  diameter  is  approximately  1  :  2. 

The  characteristic  form  of  the  bacillus  is  one  with  a 
straight  axis,  uniform  thickness  throughout,  and  flat 
ends  (Fig.  13,  page  47);  but  there  are  many  exceptions 
to  this  typical  form.  Thus  frequently  the  motile  bac- 
teria have  rounded  ends  (Fig.  10,  page  43);  many  of 
the  more  slender  forms  have  the  long  axis  bent;  some 
few  species,  such  as  the  diphtheria  bacilli  (Fig.  5),  in- 
variably produce  many  cells  whose  thickness  is  very 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      39 


unequal  at  different  portions.     Spore   formation  also 
causes  an  irregularity  of  the  cell  outline  (Fig.  12). 

The  bacilli  divide  only  in  the  plane  perpendicular  to 
their  long  axis.     A  classification,  therefore,  of  bacilli 


FIG.  5. 


FIG.  6. 


^~ —     ^-^ 

i,  single  and  in  threads. 


\&  j3fc  >  *  >  7.  \         /*V&~  OF  T 

$S£«g\  (  UNWEF 

^f|-/^|^j    V.C/Li^^ 


Small  bacilli,  mostly  in  pairs. 


according  to  their  manner  of  grouping  is  much  simpler 
than  in  the  case  of  the  cocci.  We  may  thus  have  bacilli 
as  isolated  cells,  as  pairs,  or  as  longer  or  shorter  chains. 
3.  Spiral  Form,  or  Spirillum.  The  members  of  the 
third  morphological  group  are  spiral  in  shape,  or  rather 


40 


BACTERIOLOGY. 


segments  of  a  spiral.  Here,  too,  we  have  large  and 
small,  slender  and  thick  spirals.  The  twisting  of  the 
long  axis,  which  here  lies  in  two  planes,  is  the  chief 
characteristic  of  this  group  of  bacteria.  Under  normal 


FIG.  8. 


FIG.  9. 


Medium-sized  spirilla. 


conditions  the  twisting  is  equal  throughout  the  entire 
length  of  the  cell.  The  spirilla,  like  the  bacilli,  divide 
only  in  one  direction.  A  single  cell,  a  pair,  or  the 
union  of  two  or  more  elements  may  thus  present  the 
appearance  of  a  short  segment  of  a  spiral  or  a  comma- 
shaped  form,  an  S-shaped  form,  or  a  complete  spiral 
or  corkscrew-like  form. 

Among  uncommon  morphological  peculiarities  in 
true  bacteria  may  be  mentioned  dichotomy,  or  branch 
formation — that  is,  a  side  growth  projecting  from  the 
bacterial  cell.  True  dichotomous  branching  has  occa- 
sionally been  observed  in  the  bacilli — viz.,  the  bacilli 
of  tuberculosis,  diphtheria,  and  glanders. 

Summary  of  Morphological  Forms  of  Bacteria.  1. 
Coccus,  or  micrococctis.  Spherical  or  subspherical 
forms. 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      41 

a.  Single  coccus,  grouped  irregularly. 

b.  Diplococcus,  forming  pairs. 

c.  Streptococcus,  forming  chains,  often   showing 

paired  cocci. 

d.  Tetracoccus,  forming  fours  by  division  through 

two  planes  of  space. 

e.  Sarcina,  forming  packets  of  eight  members  by 

division  through  three  planes  of  space. 

2.  Bacillus.     Oblong  or  cylindrical   forms,  having 
one  dimension   greater  than  any  other,  more  or  less 
straight,  and  never  forming  spirals. 

a.  Single  bacillus. 

b.  Diplobacillus  and  streptobacillus,  forming  twos 

or  longer  chains,  the  bacilli  attached  end  to 
end. 

c.  Filaments,  or  thread-like  growths,  in  which 

divisions  into  bacilli  of  the  normal  length 
are  not  apparent,  or  occur  irregularly  and 
transversely,  to  the  long  axis  of  the  growth. 

3.  Spirillum.     Cylindrical  and  curved  forms,  con- 
stituting complete  spirals  or  portions  of  spirals. 

The  determination  of  morphological  characters  for 
the  description  of  bacteria  should  always  be  made  from 
fully  developed  cultures;  those  which  are  too  young 
may  present,  as  already  noted,  immature  forms,  due  to 
rapid  multiplication,  while  in  old  cultures  altered  or 
degenerated  forms  may  be  observed. 

When  growth  is  obtained  upon  different  media,  varia- 
tions, especially  in  size,  may  sometimes  be  observed. 
These  differences  should  always  be  described,  together 
with  a  note  of  the  media  upon  which  they  were  devel- 
oped and  a  statement  as  to  whether  such  variation  is  a 
marked  feature  of  the  species  under  consideration. 


42  BACTERIOLOGY. 

The  conditions  of  temperature  and  of  nutrition  which 
favor  growth  are  very  various  for  different  species,  so 
that  no  fixed  temperature,  medium,  or  age  of  growth 
can  be  determined  upon  as  applicable  to  all  species. 
Morphological  descriptions  should  always  be  accom- 
panied by  a  definite  statement  of  the  age  of  the  growth, 
the  medium  from  which  it  was  obtained,  and  the  tem- 
perature at  which  it  was  developed. 

It  is  further  advisable  that  the  appearance  observed 
in  growths  developed  upon  a  solid  and  in  a  liquid 
medium  should  be  recorded. 

The  structure  of  bacterial  cells  has  recently  attracted 
considerable  attention  among  naturalists.  According  to 
Fischer  and  Migula,  the  bacterial  cells  consist  of  a  cell- 
membrane,  a  protoplasmic  layer,  and  a  central  fluid;  no 
nucleus  was  observed  by  them.  In  salt  solutions  and 
when  dried  upon  a  cover-glass  a  shrinkage  of  the  pro- 
toplasmic layer  with  partial  dissolution  of  the  cell-wall 
occurs,  due  to  the  abstraction  of  water.  This  process 
is  known  as  plasmolysis,  and  it  explains  the  occurrence 
of  the  clear,  unstained  spaces  so  frequently  seen  in 
the  stained  cover-glass  preparations  which  have  erro- 
neously been  taken  for  spores.  In  water,  or  by 
the  continued  action  of  salt  solution,  this  shrink- 
age does  not  take  place.  In  many  species  of  bacteria, 
such  as  the  diphtheria  bacilli,  there  is  observed  in  the 
interior  of  the  cells,  on  suitable  staining,  a  peculiar 
granulation,  to  which  Bab6s  has  given  the  name  of 
metachromatic  bodies,  but  which  Ernst  on  more  careful 
study  has  termed  sporagenous  granules. 

With  regard  to  the  cell  membrane,  it  should  be 
noticed  that  it  is  frequently  not  sharply  defined  and 
often  difficult  to  demonstrate.  In  many  species  of 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      43 

bacteria,  however,  commonly  known  as  capsule  bacteria, 
as  shown  in  Fig.  2,  the  cell  membrane  or  the  outer  layers 
of  the  membrane  are  so  much  thickened  that  the  bacteria 
seem  to  be  surrounded  by  a  gelatinous  envelope  or  cap- 
sule, which  is  distinguished  by  a  diminished  power  of 
staining  with  the  ordinary  aniline  dyes.  The  demonstra- 
tion of  this  capsule  may  be  of  help  in  differentiating 
between  certain  bacteria — e.  g.,  some  forms  of  the 
streptococcus  and  pneumococcus.  A  peculiarity  of 

FIG.  10. 


Faintly  stained  flagella  attached  to  heavily  stained  bacilli. 

the  capsule  bacteria  is  that,  except  very  rarely,  they 
exhibit  this  envelope  only  when  grown  in  the  animal 
body  or  in  special' culture  media,  such  as  liquid  blood 
serum,  bronchial  mucus,  etc.  ;  grown  on  nutrient 
gelatin,  agar,  or  potato  the  capsule  is  only  visible  under 
very  exceptional  conditions,  and  then  not  distinctly. 

The  outer  surface  of  bacteria  when  occurring  in  the 
form  of  spheres  and  short  rods  is  almost  always  smooth 


44  BACTERIOLOGY. 

and  devoid  of  appendages  ;  but  the  longer  rods  and 
spirals  are  usually  provided  with  fine  hair-like  append- 
ages or  flagella,  which  are  their  organs  of  motility. 
These  flagella,  either  singly  or  in  numbers,  are  some- 
times distributed  over  the  entire  body  of  the  cell,  or 
they  may  form  a  tuft  at  one  end  of  the  rod,  or  only 
one  polar  flagellum  is  found.  The  polar  flagella 
appear  on  the  bacteria  shortly  before  division.  The 
nature  of  flagella  is  little  understood;  they  are  believed 
by  some  to  be  formed  of  protoplasmic  material  which 
penetrates  the  cell  membrane,  and  probably  have  the 
property  of  protrusion  and  retraction.  So  far  as  we 
know,  the  flagella  are  the  only  means  of  locomotion 
possessed  by  the  bacteria.  They  are  not  readily 
stained,  special  staining  agents  being  required  for 
this  purpose.  The  envelope  of  the  bacteria,  which 
usually  remains  unstained  with  the  ordinary  dyes, 
then  becomes  colored  and  more  distinctly  visible  than 
is  commonly  the  case.  Occasionally,  however,  some 
portion  of  the  envelope  remains  unstained,  when  the 
flagella  present  the  appearance  of  being  detached  from 
the  body  of  the  bacteria  by  a  narrow  zone.  Unfor- 
tunately, many  of  the  methods  employed  for  staining 
flagella  cause  them  to  become  degenerated,  so  that  their 
perfect  demonstration  is  often  very  difficult.  In  cul- 
tures of  richly  flagellated  bacteria  peculiar  pleated 
masses  sometimes  are  observed,  consisting  of  flagella 
which  have  been  detached  and  then  matted  together. 
Bacteria  may  lose  their  power  of  producing  flagella  for 
a  series  of  generations.  Whether  their  power  be  per- 
manently lost  or  not  we  do  not  know. 

The  vegetative  reproduction  of  bacteria  takes  place  by 
division.     When  development  is  in  progress  a  single 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      45 

cell  will  be  seen  to  elongate,  in  the  case  of  spherical 
bacteria  only  slightly,  and  in  the  rod-shaped  organ- 
isms considerably  in  one  direction.  Over  the  centre 
of  the  long  axis  thus  formed  will  appear  a  slight  in- 
dentation in  the  outer  envelope  of  the  cell ;  this  inden- 
tation increases  in  extent  until  there  exists  eventually 
two  individuals.  As  a  rule,  the  cells  separate  from 
one  another  soon  after  division,  but  occasionally  they 
remain  together  for  a  time,  forming  pairs  and  chains. 
Under  certain  conditions  of  nutrition  long  threads  or 
filaments  are  formed,  whirh,  however,  when  put  in 
contact  with  new  food,  break  up  into  fragments.  At 
times,  when  the  culture  media  are  exhausted  or  nearly 
so,  the  bacilli  and  spirilla  will  be  found  to  go  on  divid- 
ing, with  little  or  no  increase  in  length,  and  thus  coc- 
cus-like forms  result  ;  but  when  these  are  given  fresh 
food  under  suitable  conditions  they  elongate  and  repro- 
duce the  usual  shaped  organisms.  According  to  recent 
investigations  on  the  subject  of  cell  reproduction,  the 
division  of  the  cell  starts  from  the  protoplasmic  layer, 
the  central  space  being  passively  destroyed,  and  the 
outer  envelop  is  only  secondarily  concerned  in  the 
process.  This  would  indicate  that  the  central  space 
is  not  a  true  nucleus,  otherwise  the  division  of  the 
nucleus  should  precede  the  cell  division.  The  com- 
plete process  of  cell  reproduction  in  most  varieties 
occupies,  under  favorable  conditions,  about  twenty  to 
thirty  minutes. 

But  although  elongation  in  the  greater  diameter 
and  transverse  division  is  the  rule  for  the  majority  of 
bacteria,  there  are  certain  groups,  as  the  sarcinse,  for 
example,  which  divide  more  or  less  regularly  in  three 
directions.  Instead  of  becoming  separated  from  each 


46  BACTERIOLOGY. 

other  as  single  cells,  the  tendency  then  is  for  the  seg- 
mentation to  be  incomplete,  the  cells  remaining  together 
in  masses.  The  indentations  upon  these  masses  or 
cubes,  which  indicate  the  point  of  incomplete  fission, 
give  to  these  bundles  of  cells  the  appearance  commonly 
ascribed  to  them — that  of  a  bale  of  rags.  As  already 
said,  division  in  two  opposite  directions  results  in  the 
formation  of  a  group  of  forms  as  tetrads.  Division 
irregularly  in  all  directions  results  in  the  production 
of  clusters.  The  rod-shaped  bacteria  never  divide 
longitudinally. 

Spore-formation  must  be  distinguished  from  vegeta- 
tive reproduction.  This  is  the  process  by  which  the 
organisms  are  enabled  to  enter  a  stage  in  which  they 
resist  deleterious  influences  to  a  much  higher  degree 
than  is  possible  for  them  in  the  growing  or  vegetative 
condition.  There  are  two  kinds  of  spores  which  have 
been  described :  1.  Endospores,  which  are  strongly 
refractile  and  glistening  in  appearance,  oval  or  round 
in  shape,  and  developed  within  the  interior  of  the  cell. 
They  are  characterized  by  the  power  of  resisting  to  a 
considerable  extent  the  injurious  influences  of  heat, 
desiccation,  and  chemical  disinfectants.  2.  Arthro- 
sporeSy  or  jointed  spores,  developed  not  within  the  cell 
but  as  a  sprout  like  separation  of  one  of  its  extremi- 
ties. These  jointed  bodies  are  believed  by  some  to 
have  also  greater  resisting  power  to  desiccation,  etc., 
than  the  ordinary  cells,  though  less  than  the  endo- 
spores,  and  to  serve  the  purpose  of  reproductive 
elements.  Recent  researches  into  the  formation  of 
arthrospores,  however,  have  resulted  in  nothing  defi- 
nite, and  the  question  of  their  existence  even  in  bac- 
teria still  remains  open.  In  describing  the  biological 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      47 


characters,  therefore,  of  the  various  organisms  whenever 
spores  are  mentioned,  it  will  be  understood  that  only 
the  endogenous  spores  are  meant. 


FIG.  11. 


Unstained  spores  in  slightly  distended  bacilli.    (The  spores  are  the  light 
spots  in  heavily  stained  bacilli.) 


FIG.  12. 


FIG.  13. 


Spores  in  distended  ends  01 
bacilli. 


Unstained  spores  in  centre  of  bacilli 
arranged  in  chains. 


The  production  of  endospores  in  the  different  species 
of  bacteria,  though  not  identical,  is  very  similar.  To 
observe  the  formation  of  spores  in  any  species  it  is  best 
to  employ  a  streak  culture  on  nutrient  agar  or  a  potato 
culture,  which  should  be  kept  at  the  temperature  nearest 


48  BACTERIOLOGY. 

the  optimum  of  the  organism  to  be  examined.  If  at  the 
end  of  twelve,  eighteen,  twenty-four,  thirty,  thirty-six 
hours,  etc.,  specimens  of  the  culture  are  observed  first 
unstained  in  the  hanging  drops,  and  then,  if  round  or 
oval,  highly  refractile  bodies  are  seen,  they  should  be 
stained  for  spores. 

According  to  Fischer  motile  bacteria  always  come  to 
a  state  of  rest  or  immobility  previous  to  spore-forma- 
tion. Several  species  first  become  elongated.  The 
anthrax  bacillus  does  this,  and  a  description  of  the 
method  of  its  production  of  spores  may  serve  as  an 
illustration  of  the  process  In  the  beginning  the  pro- 
toplasm of  the  elongated  filaments  is  homogeneous, 
but  after  a  time  it  becomes  turbid  and  finely  granular. 
These  fine  granules  are  then  replaced  by  a  smaller 
number  of  coarser  granules,  which  are  finally  amalga- 
mated into  a  spherical  or  oval  refractile  body.  This 
is  the  spore.  As  soon  as  the  process  is  completed  there 
appears  between  two  spores  a  delicate  partition  wall. 
For  a  time  the  spores  are  retained  in  a  linear  position 
by  the  cell  membrane  of  the  bacillus,  but  this  is  later 
dissolved  or  broken  up  and  the  spores  are  set  free. 
Not  all  the  cells  that  make  the  effort  to  form  spores, 
as  shown  by  the  spherical  bodies  contained  in  them, 
bring  these  to  maturity;  indeed,  many  varieties,  under 
certain  cultural  conditions,  lose  their  property  of  forming 
spores.  The  following  are  the  most  important  spore 
types  :  (a)  The  spore  lying  in  the  interior  of  a  short 
undistended  cell  ;  (6)  the  spore  lying  in  the  interior  of 
a  short  undistended  cell  forming  one  of  the  elements  of 
a  long  filament  ;  (c)  the  spore  lying  at  the  extremity 
of  an  undistended  cell  much  enlarged  at  that  end — the 
so-called  "  head  spore  ;"  and  (d)  the  spore  lying  in 


GENERAL  CHARACTERISTICS  OF  BACTERIA.      49 

the  interior  of  a  cell  very  much  enlarged  in  its  central 
portion,  giving  it  a  spindle  shape. 

The  germination  of  spores  takes  place  as  follows  : 
By  the  absorption  of  water  they  become  swollen  and 
pale  in  color,  losing  their  shining,  refractile  appear- 
ance. Later  a  little  protuberance  is  seen  upon  one  side 
or  at  one  extremity  of  the  spore,  and  this  rapidly 
grows  out  to  form  a  rod  which  consists  of  soft-growing 
protoplasm  enveloped  in  a  membrane  which  is  formed 
of  the  endosporium  or  inner  layer  of  the  cellular  en- 
velope of  the  spore  The  outer  envelope,  or  exosporium, 
is  cast  off,  and  may  be  seen  in  the  vicinity  of  the 
newly-formed  rod.  Sometimes  the  vegetative  cell 
emerges  from  one  extremity  of  the  oval  spore,  and 
in  other  species  the  exosporium  is  ruptured  and  the 
bacillus  emerges  from  the  side. 

In  old  cultures  of  bacteria,  where  the  deleterious  sub- 
stances have  developed  and  the  food-stuffs  have  been 
largely  used,  there  are  frequently  found  very  irregular 
or  distorted  forms,  due  to  the  abnormal  development  and 
division  of  the  bacterial  cells  under  the  unfavorable 
conditions  present.  These  are  spoken  of  as  involution 
or  degenerated  forms.  If  these  deformed  cells  are  placed 
under  suitable  conditions  they  produce  again  normally 
fashioned  organisms. 


CHAPTER  II. 

THE      CHEMICAL     COMPOSITION     OF      BACTERIA — THE 
CONDITIONS    SUITABLE     FOR    THEIR    GROWTH. 

Chemical  Composition.  Qualitatively  considered,  the 
bodies  of  bacteria  consist  largely  of  water,  salts,  fats, 
and  albuminous  substances.  There  are  also  present, 
in  smaller  quantities,  extractive  substances  soluble  in 
alcohol  and  in  ether.  According  to  Cramer,  there  is 
no  grape-sugar  found  in  any  bacterial  species,  but  many 
bacteria  contain  amyloid  substances  which  give  a  blue 
reaction  with  iodine.  True  cellulose  has  been  found  in 
the  bacillus  subtilis  and  an  organism  closely  allied  to 
the  bacillus  coli;  the  tubercle  bacillus  also  forms  cellu- 
lose in  the  animal  body,  though  no  cellulose  has  been 
found  in  cultures  of  the  tubercle  bacillus.  But  from 
these  and  from  cultures  of  a  "  capsule  bacillus  from 
water/7  allied  to  the  pneumococcus  of  Friedlander, 
large  quantities  of  a  gelatinous  carbohydrate  similar 
to  hemi-cellulose  have  been  obtained.  Nuclein,  first 
demonstrated  by  Vanderville,  is  only  found  with  diffi- 
culty; but  the  nuclein  bases — xanthin,  guanin,  and 
adenin — have  been  found  in  considerable  amounts. 
There  is  a  group  of  bacteria  which  contain  sulphur — 
viz.,  the  begyiatoa — and  another  group,  the  cladothrix, 
is  capable  of  separating  ferric  oxide  from  water  con- 
taining iron. 

Some  light  has  been  thrown  upon  the  chemical  com- 


CHEMICAL  COMPOSITION  OF  BACTERIA.       51 

position  of  bacteria,  quantitatively,  by  the  studies  of 
Cramer,  though  so  far  only  a  few  species  have  been 
thoroughly  investigated.  The  percentage  of  water 
contained  in  bacteria  grown  on  solid  culture  media,  as 
well  as  the  amount  of  ash,  depend  largely  on  the 
composition  of  the  media.  Thus  the  bacillus  prodigio- 
sus  when  grown  on  potato  contains  21.5  per  cent,  of 
dry  residue  and  2.7  per  cent,  of  ash  ;  when  cultivated 
on  turnips  it  contains  12.6  per  cent,  of  dry  residue  and 
1.3  per  cent,  of  ash.  Beside  the  concentration  of  the 
culture,  its  temperature  and  age  also  influence  the 
amount  of  residue  and  ash  produced.  The  residue 
varies,  moreover,  in  its  composition  in  the  same  species 
under  the  influence  of  the  culture  media  employed. 
Thus  the  Friedlander  pneumonia  bacillus  grown  on 
nutrient  agar  containing  peptone  yields  of  residue : 

With  1  per  cent.  With  5  per  cent. 

peptone.  peptone. 

Nitrogenous  matter        .     71.7  per  ct.          79.8  per  ct. 
Extractives    .         .         .     10.3         "  11.3         " 

Ash        ....     13.9         "  10.3         " 

With  1  per  ct.  peptone 

+  5  per  ct.  glucose. 
Nitrogenous  matter  ....     63.6  per  ct. 

Extractives      .         .         .         .         .         .22.7         " 

Ash .       7.8         " 

It  would  thus  appear  that  an  additional  quantity  of 
peptone  in  the  culture  media  tends  to  increase  the  per- 
centage of  nitrogenous  matter  in  the  bacillus,  while 
the  addition  of  glucose  decreases  it. 

The  cholera  spirillum  shows  still  greater  variations 
in  the  residue  when  grown  in  soda  bouillon  containing 
albumin  than  in  Uschinsky's  medium,  which  is  free 
from  albumin.  Thus  Cramer  found  as  an  average 


52  BACTERIOLOGY. 

yield  of  residue  from  five  different  varieties  of  cholera 
spirilla  :  Albumin  65  per  cent,  and  ash  31  per  cent, 
when  grown  in  soda  bouillon,  while  in  Uschinsky's 
solution  there  was  only  45  per  cent,  albumin  and  11 
per  cent.  ash.  The  five  varieties  of  spirilla  which  in 
soda  bouillon  yielded  almost  exactly  the  same  quanti- 
ties of  albumin  and  ash,  in  the  other  medium  free  from 
albumin  exhibited  a  very  variable  composition.  This 
shows  how  little  dependence  can  be  placed  upon  any 
single  chemical  or  cultural  reaction  for  the  differentia- 
tion of  two  species  of  bacteria.  Judging  from  the  per- 
centage composition  of  the  cholera  spirilla  when  grown 
in  the  Uschinsky  medium,  these  five  varieties,  taken 
from  Paris,  Hamburg,  Shanghai,  etc.,  might  be  con- 
sidered to  be  different  species,  whereas  they  were  prob- 
ably merely  varieties  of  the  same  species  of  bacteria. 

CONDITIONS  OF   GROWTH. 

Culture  Media.  Although  there  are  among  the  bac- 
teria related  to  disease  a  number  which  are  met  with 
only  in  the  bodies  of  living  animals  or  plants,  and, 
therefore,  so  far  as  we  know,  strictly  parasites,  yet 
most  pathogenic  bacteria  can  be  cultivated  more  or  less 
readily  in  artificial  culture  media  under  suitable  con- 
ditions, as,  for  example,  the  tubercle  bacillus  and  the 
gonococcus.  The  majority  of  bacteria  which  occur 
usually  as  saprophytes  are  easily  cultivated  artificially; 
but  there  are  some,  such  as  various 'micro-organisms 
found  in  the  saliva  and  in  water,  which,  with  our 
present  knowledge,  are  either  difficult  or  impossible  to 
cultivate. 

All  bacterial  culture  media  must  contain  an  abun- 


CONDITIONS  OF  GROWTH.  53 

dance  of  water  ;  salts  are  also  indispensable,  and  there 
must  be  organic  material  as  a  source  of  carbon  and 
nitrogen.  The  greater  number  of  important  bacteria 
and  all  the  pathogenic  species  thrive  best  in  media 
containing  albuminoid  substances  and  of  a  slightly 
alkaline  reaction.  The  demands  of  bacteria  in  the 
composition  of  the  culture  media  vary  very  consid- 
erably. There  are  some  species  of  water  bacteria, 
for  instance,  which  require  so  little  organic  material 
that  they  will  grow  in  water  that  has  been  twice 
distilled.  In  such  cases  development  probably  takes 
place  owing  to  some  contamination  of  the  water,  or 
else  through  the  decomposition  of  the  ammonia  and 
carbonic  acid  in  the  air.  A  certain  species  will 
grow  abundantly  in  water  containing  ammonium  car- 
bonate in  solution  and  no  other  source  of  carbon  and 
nitrogen.  This  shows  the  power  of  some  bacteria  of 
producing  cell  substance  from  the  simplest  materials — a 
power  which  belongs  to  the  higher  plants  which  obtain 
their  nourishment  from  the  air  through  their  chlorophyl 
and  the  assistance  of  sunlight.  Few  bacteria,  how- 
ever, of  any  importance  in  medicine  are  so  easily  satis- 
fied, though  there  are  many  species  which  are  able 
to  develop  without  the  presence  of  albumin  and  in 
comparatively  simple  culture  media,  such  as  the  culture 
liquid  proposed  by  Uschinsky,  or  the  simpler  one  of 
Voges  and  Fraenkel,  which  consists  of:  Water,  1000; 
sodium  chloride,  5  ;  neutral  sodium  phosphate,  2  ; 
ammonium  acetate,  6;  and  asparagin,  4.  In  these 
media  many  bacteria  grow  well. 

When  we  consider  in  detail  the  source  of  the  more 
important  chemical  ingredients  of  bacteria  we  find  that 
their  nitrogen  is  most  readily  obtained  from  diffusible 


54  BACTERIOLOGY. 

abuminoid  material  and  less  easily  from  ammonium 
compounds.  Their  carbon  they  derive  from  albumin, 
peptone,  sugar  and  other  allied  carbohydrates,  glycerin, 
fats,  and  other  organic  substances.  It  is  an  interest- 
ing fact,  that  even  compounds  which  in  considerable 
concentration  are  extremely  poisonous,  can,  when  in 
sufficient  dilution,  provide  the  necessary  carbon;  thus 
some  derive  it  from  carbolic  acid  in  very  dilute  solu- 
tions. Another  species  of  bacteria  isolated  by  Wino- 
gradsky  were  shown  by  him  to  derive  their  carbon 
from  CO2. 

The  value  of  substances  as  a  source  of  .nutrition  is 
often  influenced  by  the  presence  of  other  materials,  as, 
for  instance,  the  value  of  asparagin  is  increased  by  the 
presence  of  sugars.  Further,  material  from  which 
nitrogen  and  carbon  cannot  be  directly  obtained  still 
become  assimilable  after  being  subjected  to  the  influ- 
ence of  bacterial  ferments.  The  profound  and  diverse 
changes  produced  by  the  different  ferments  make  it 
almost  impossible  to  establish,  except  in  the  most  gen- 
eral way,  the  nutritive  value  of  any  mixture  for  a  large 
number  of  bacteria  through  a  simple  knowledge  of  its 
chemical  composition.  The  special  culture  media,  such 
as  bouillon,  blood-serum,  etc.,  for  the  development  of 
bacteria  will  be  dealt  with  in  a  later  chapter. 

The  relation  of  bacteria  to  oxygen:  The  majority  abso- 
lutely require  oxygen  for  their  growth,  but  a  consider- 
able minority  fail  to  grow  unless  it  is  excluded.  A 
knowledge  of  this  latter  fact  we  owe  to  Pasteur,  who 
divided  bacteria  into  aerobic  and  anaerobic.  Between 
these  two  groups  we  have  those  that  can  grow  either 
with  or  without  the  access  of  oxygen. 

Some  at  least  of  the  strict  anaerobic  bacteria  require 


CONDITIONS  OF  GROWTH.  55 

for  the  full  development  of  their  life  functions  the 
presence  of  fermentable  substances,  such  as  sugars, 
from  which  they  obtain  oxygen.  Among  bacteria  can 
be  found  all  gradations  between  those  bacteria  which 
develop  only  in  the  presence  of  oxygen  to  those  which 
develop  only  in  its  absence.  In  so  far  as  for  any 
variety  the  amount  of  oxygen  present  is  unfavorable 
there  will  be  more  or  less  restriction  in  some  of  the  life 
processes  of  these  bacteria,  such  as  pigment  and  toxin 
production,  spore  formation,  etc.  It  has  also  been 
found  that  some,  at  least,  of  the  aerobic  bacteria  can 
be  accustomed  to  grow  without  oxygen,  and  that  some 
of  the  anaerobics  can  be  accustomed  to  grow  with  it. 

Sulphur  and  phosphorus  are  two  important  food-stuffs 
required  by  bacteria.  Either  calcium  or  magnesium 
and  sodium  or  potassium  are  also  usually  required  for 
bacterial  growth.  Iron  is  demanded  by  but  few  varie- 
ties. 

When  we  consider  the  more  complex  culture  media, 
either  those  naturally  existing,  such  as  blood-serum, 
or  those  created  by  us  for  the  cultivation  of  bacteria, 
we  find,  beyond  the  necessary  amount  of  soluble  food- 
stuffs, that  the  relative  proportion  of  each  form  and 
the  total  concentration  are  of  great  importance.  It  is, 
nevertheless,  true  that  very  wide  differences  can  exist 
with  but  slight  effect  upon  the  development  of  bacteria, 
the  development  of  the  bacteria  usually  ceasing  through 
the  accumulation  of  deleterious  substances  in  the  cul- 
ture media  rather  than  through  food  exhaustion. 

The  reaction  of  the  nutritive  media  is  of  very  great 
importance.  Most  bacteria  grow  best  in  those  that  are 
slightly  alkaline  or  neutral.  Only  a  few  varieties  require 
an  acid  medium,  and  none  of  these  belong  to  the  parasitic 


56  BACTERIOLOGY. 

bacteria.  An  amount  of  acid  or  alkali  insufficient  to 
prevent  the  development  of  bacteria  may  still  suffice  to 
rob  them  of  some  of  their  most  important  functions, 
such  as  the  production  of  poison.  The  different  effect 
upon  closely  allied  varieties  of  bacteria  of  a  slight 
excess  of  acid  or  alkali  is  sometimes  made  use  of  in 
separating  those  which  may  be  closely  allied  in  many 
other  respects. 

The  influence  of  one  species  upon  the  growth  of 
another,  either  when  the  bacteria  grow  together  or 
follow  one  another,  is  very  marked.  The  development 
of  one  variety  of  bacteria  in  a  medium  causes  that  sub- 
stance, in  the  majority  of  instances,  to  become  less  suit- 
able for  the  growth  of  other  bacteria.  This  isxdue 
partly  to  the  impoverishment  of  the  food-stuff,  but 
more  to  the  production  of  chemical  substances  or 
enzymes,  which  are  antagonistic  not  only  to  the  growth 
of  the  bacteria  producing  them,  but  to  many  other 
varieties  also;  less  frequently  the  changes  produced  by 
one  variety  of  bacteria  in  the  food-stuff  are  favorable 
for  some  other  form. 

For  the  growth  of  bacteria  a  suitable  temperature  is 
absolutely  requisite.  For  different  varieties  the  most 
favorable  temperature  varies,  but  for  all  a  range  of 
about  2J°  C.  above  or  below  this  most  favorable  point 
covers  the  limits  for  their  most  vigorous  growth.  Few 
bacteria  grow  well  under  10°  C.  and  few  over  40°  C. ; 
2°  C.  is  about  the  lowest  temperature  that  any  bacteria 
have  been  found  to  grow  and  70°  C.  the  highest. 

In  many  instances  the  temperature  of  the  soil  in 
which  the  bacteria  are  deposited  is  the  controlling 
factor  in  deciding  whether  growth  will  or  will  not  take 
place.  Thus  nearly  all  parasitic  hacleria  require  a 


CONDITIONS  OF  GROWTH.  57 

temperature  near  that  of  the  body  for  their  develop- 
ment, while  many  saprophytic  bacteria  can  grow  only 
at  much  lower  temperatures.  Bacteria  when  exposed 
to  lower  temperature  than  suffices  for  their  growth, 
while  having  their  activities  decreased,  are  not  other- 
wise injured;  while  exposure  to  higher  temperatures 
than  allows  of  growth  destroys  the  life  of  the  bacteria. 
The  relations  of  the  temperature  to  bacterial  life  and 
death  will  be  dealt  with  fully  in  a  later  chapter. 


CHAPTER   III. 

VITAL     PHENOMENA     OF     BACTERFA — MOTION,     HEAT 
AND    LIGHT    PRODUCTION — CHEMICAL    EFFECTS. 

Motility.  Many  bacteria  when  examined  under  the 
microscope  are  seen  to  exhibit  active  movements  in 
fluids.  This  motility  is  produced  by  the  fine  hair-like 
flagella  attached  to  all  motile  species.  The  movements 
are  of  a  varying  character,  being  described  as  creeping, 
waddling,  rotary,  undulatory,  sinuous,  snake-like,  etc. 
At  one  time  they  may  be  slow  and  sluggish,  at  another 
so  rapid  that  any  detailed  observation  is  impossible. 
Some  bacteria  are  very  active  in  their  movements,  dif- 
ferent individuals  progressing  rapidly  in  different  direc- 
tions, while  with  many  it  is  difficult  to  say  positively 
whether  there  is  any  actual  motility  or  whether  the 
organism  shows  only  molecular  movements — so-called 
"  Brownian  "  movements — a  dancing,  trembling  mo- 
tion possessed  by  all  finely  divided  organic  particles. 
If  in  doubt  in  such  cases  it  is  best,  wkeVe  the  matter  is 
of  importance  and  one  is  skilled  in  the  technique  of 
staining,  to  stain  the  organisms  for  flagella,  and  also  to 
examine  them  in  a  0.1  per  cent,  bichloride  of  mercury 
solution,  when,  if  the  movements  continue,  they  are 
purely  molecular.  Not  all  species  of  bacteria  which 
have  flagella,  however,  exhibit  at  all  times  spontaneous 
movements;  such  movements  may  be  absent  in  certain 
culture  media  and  at  too  low  or  too  high  temperatures, 
or  of  either  an  insufficient  or  excessive  supply  of  oxygen. 


VITAL  PHENOMENA  OF  BACTERIA.  59 

Some  chemical  substances  seem  to  exert  a  peculiar 
attraction  for  bacteria,  known  as  positive  chemotaxis, 
while  others  repel  them — negative  chemotaxis.  More- 
over, all  varieties  are  not  affected  alike,  for  the  same 
substances  may  exert  on  some  bacteria  an  attraction 
and  on  others  a  repulsion.  Oxygen,  for  example, 
attracts  aerobic  and  repels  anaerobic  bacteria,  and  for 
each  variety  there  is  a  definite  proportion  of  oxygen, 
which  most  strongly  attracts.  The  chernotaxic  prop- 
erties of  substances  are  tested  by  pushing  the  open  end 
of  a  fine  capillary  tube,  filled  with  the  substance  to  be 
tested,  into  the  edge  of  a  drop  of  culture  fluid  contain- 
ing bacteria  and  examining  the  hanging  drop  under 
the  microscope.  We  are  able  thus  to  watch  the  action  of 
the  bacteria  and  note  whether  they  crowd  about  the  tube 
opening  or  are  repelled  from  it.  Substances  showing 
positive  chemotaxis  for  nearly  all  bacteria  are  peptone, 
urea,  and  very  weak  solutions  of  bichloride  of  mercury. 
While  among  those  showing  negative  chemotaxis  are 
alcohol  and  many  of  the  metallic  salts. 

The  Production  of  Light.  Bacteria  which  have  the 
property  of  emitting  light  are  quite  widely  distributed 
in  nature,  particularly  in  media  rich  in  salt,  as  in  sea- 
water,  salt  fish,  etc.  Many  of  these,  chiefly  bacilli  and 
spirilla,  have  been  accurately  studied.  The  emission  of 
light  is  a  property  of  the  living  protoplasm  of  the  bac- 
teria, and  is  not  usually  due  to  the  oxidation  of  any  pho- 
togenic substance  given  off  by  them ;  at  least  only  in  two 
instances  has  such  substance  been  claimed  to  have  been 
isolated.  Every  agent  which  is  injurious  to  the  exist- 
ence of  the  bacteria  affects  this  property.  Thus,  cold 
paralyzes  them  and  interrupts  their  power  of  emit- 
ting light.  High  temperature,  acids,  chloroform,  etc., 


60  BACTERIOLOGY. 

inhibit  for  a  time  or  destroy  this  property.  Living  bac- 
teria are  always  found  in  phosphorescent  cultures;  a 
filtered  culture  free  from  germs  is  invariably  non-phos- 
phorescent; but  while  the  organism  cannot  emit  light  ex- 
cept during  life,  it  can  live  without  emitting  light,  as  in 
an  atmosphere  of  carbonic  acid  gas,  for  instance.  Most 
organisms  require,  in  order  to  be  able  to  emit  light,  the 
presence  of  peptone  and  oxygen,  and  many  also  need 
carbon  and  nitrogen.  They  are  best  grown  under  free 
access  of  oxygen  in  a  culture  medium  prepared  by 
boiling  fish  in  sea- water  (or  water  containing  3  per 
cent,  sea-salt),  to  which  1  per  cent,  peptone,  1  per 
cent,  glycerin,  and  0.5  per  cent,  asparagin  are  added. 
Even  in  this  medium  the  power  of  emitting  light  is 
soon  lost  unless  the  organism  is  constantly  trans- 
planted to  fresh  m^dia. 

Thermic  Effects.  The  production  of  heat  by  bacteria 
does  not  attract  attention  in  our  usual  cultures  because 
of  its  slight  amount,  and  even  fermenting  culture 
liquids  with  abundance  of  bacteria  cause  no  sensa- 
tion of  warmth  when  touched  by  the  hand.  Careful 
tests,  however,  show  that  heat  is  produced.  The 
increase  of  temperature  in  organic  substances  when 
stored  in  a  moist  condition,  as  tobacco,  hay,  manure, 
etc.,  is  one  partly  at  least  due  to  the  action  of  bacteria. 
Rabinowitsch  suggests  that  very  probably  the  high  tem- 
perature which  is  here  exhibited  is  caused  in  part  by  the 
so-called  thermophilic  bacteria,  but  there  are  no  accurate 
observations  as  to  the  true  source  of  this  heat. 

Chemical  Effects.  The  processes  which  bodies  being 
split  up  undergo  depend,  first,  on  the  chemical  nature 
of  the  bodies  involved  and  the  conditions  under  which 
they  exist,  and,  secondly,  on  the  varieties  of  bacteria 


VITAL  PHENOMENA  OF  BACTERIA.  61 

present.  A  complete  description  of  these  chemical 
changes  is  at  present  impossible.  Chemists  can  as  yet 
only  enumerate  some  of  the  final  substances  evolved, 
and  describe,  in  a  few  cases,  the  manner  in  which  they 
were  produced.  Bacteria  are  able  to  construct  their 
body  substance  out  of  various  kinds  of  nutrient  mate- 
rials and  also  to  produce  fermentation  products  or 
poisons,  and  they  are  able  to  do  these  things  either 
analytically  or  synthetically  with  almost  equal  ease. 
This  ambidextrous  metabolic  power  exists,  according 
to  Hueppe,  among  bacteria  to  an  extent  known  as  yet 
among  no  other  living  things. 

In  the  chemical  building  up  of  their  body  substance 
we  can  distinguish,  as  Hueppe  concisely  puts  it,  several 
groups  of  phenomena:  Polymerization,  a  sort  of  doub- 
ling up  of  a  simple  compound;  synthesis,  a  union  of 
different  kinds  of  simple  compounds  into  one  or  more 
complex  substances;  formation  of  anhydride,  by  which 
new  substances  arise  from  a  compound  through  the  loss 
of  water;  and  reduction  or  loss  of  oxygen,  which  is 
brought  about  especially  by  the  entrance  of  hydrogen 
into  the  molecule.  The  breaking  down  of  organic 
bodies  of  complicated  molecular  structure  into  simpler 
combinations  takes  place,  on  the  other  hand,  through 
the  loosening  of  the  bonds  of  polymerization;  through 
hyd ration  or  entrance  of  water  into  the  molecule,  and 
through  oxidation. 

The  chemical  effects  which  take  place  from  the  action 
of  bacteria  are  greatly  influenced  by  the  presence  or 
absence  of  free  oxygen.  The  access  of  pure  atmos- 
pheric oxygen  makes  the  life  processes  of  most  bacteria 
more  easy,  but  is  not  indispensable  when  available 
substances  are  present  which  can  be  broken  up  with 


62  BACTERIOLOGY. 

sufficient  ease.  The  standard  of  availability  is  very 
different  for  different  bacteria.  Life  processes  carried 
on  without  oxygen  do  not  effect  any  profound  molecular 
changes  in  the  organic  material  which  is  broken  up; 
but  in  order  that  the  living  organism  may  obtain  the 
requisite  quantities  of  energy  from  this  mode  of  life, 
a  proportionately  large  amount  of  material  must  be 
superficially  disintegrated.  Therein  lies  the  mechan- 
ical foundation  for  the  power  of  a  small  amount  of  fer- 
ment to  cause  the  production  of  much  alcohol  or  lactic 
acid,  and  that  parasites  which  have  invaded  the  living 
body  can  generate  intensely  poisonous  substances  out 
of  the  body  proteids. 

In  the  presence  of  oxygen  the  decomposition  products 
that  are  formed  by  the  attack  of  the  anaerobic  bacteria 
are  further  decomposed  and  oxidized  by  the  aerobes; 
they  are  thereby  rendered,  as  a  rule,  inert,  and  conse- 
quently harmless.  Some  bacteria  have  adapted  them- 
selves to  the  exclusive  use  of  compound  oxygen,  using 
those  compounds  from  which  oxygen  can  be  obtained, 
and  others — the  obligatory  anaerobes — are  able  to  live 
only  in  the  presence  of  free  oxygen,  The  facts  of  anae'ro- 
biosis  are  of  great  importance  to  technical  biology  and 
to  pathology.  Since,  under  strictly  anaerobic  conditions, 
any  secondary  oxidation  of  the  primary  decomposition 
products  is  impossible,  the  latter  accumulate  without 
formation  of  by-products.  Many  parasitic  bacteria  are 
found  to  produce  far  more  poison  in  the  absence  of  air 
than  in  its  presence. 

Organized  and  Unorganized  Ferments.  All  the  chem- 
ical effects  of  bacteria  are  largely  dependent  upon  the 
composition  of  the  culture  media.  Thus  many  species 
of  bacteria  which  in  albuminous  media  produce  no 


VITAL  PHENOMENA  OF  BACTERIA.  63 

visible  change,  when  sugar  is  added  decompose  it,  with 
the  production  of  gas.  The  term  fermentation  is  dif- 
ferently used  by  different  authors.  Some  call  every 
kind  of  decomposition  due  to  bacteria  a  fermentation, 
speaking  thus  of  the  putrefactive  fermentation  of  albu- 
minous substances;  others  limit  the  term  to  the  process 
when  accompanied  by  the  visible  production  of  gas; 
others,  again,  take  fermentation  to  mean  only  the 
decomposition  of  carbohydrates,  with  or  without  gas- 
production. 

Fermentation  may  be  defined  as  a  chemical  decom- 
position of  an  organic  compound,  induced  by  living 
organisms  or  substances  contained  within  them  (organ- 
ized ferments),  or  by  chemical  substances  thrown  off 
from  the  bacteria  (unorganized  or  chemical  ferments 
or  enzymes).  In  the  first  the  action  is  due  to  the 
growth  of  the  organisms  producing  the  ferment,1  as 
in  the  formation  of  acetic  acid  from  alcohol  by  the 
action  of  the  vinegar-plant,  and  in  the  second  the 
enzyme  causes  a  structural  change  without  losing  its 
identity,  as  in  digestion.  These  enzymes  even  when 
present  in  the  most  minute  quantities  have  the  power 
of  splitting  up  or  decomposing  complex  organic  com- 
pounds into  simpler,  more  easily  soluble  and  diffu- 
sible molecules.  We  can  only  speak  of  chemical  fer- 
ments when  it  can  be  demonstrated  that  the  fermenta- 
tion continues  in  the  absence  of  all  living  bacteria. 

1  Buchner  (Berichte  d.  deutsch.  chem.  Gesellsch.,  xxx.  117-124  and  1110- 
1113)  has  shown  that  even  in  those  cases  of  fermentation  in  which,  until 
lately,  we  have  believed  the  organized  cell  itself  was  necessarily  concerned 
that  the  cell  protoplasm  squeezed  from  its  capsule  is  able  to  cause  the  same 
changes  as  the  organized  cells.  This  brings  fermentation  by  unorganized  and 
organized  ferments  very  closely  together,  the  one  being  a  substance  thrown 
off  from  the  cell,  the  other  a  substance  ordinarily  retained  in  the  cell.  The 
increase  of  both  ceases  with  the  death  of  the  bacteria  producing  them. 


64  BACTERIOLOGY. 

This  may  be  accomplished  by  the  addition  of  disinfect- 
ants— carbolic  acid,  chloroform,  ether,  etc. — to  the  cul- 
tures or  by  filtration.  Ferments,  like  albuminoids, 
are  non-dialyzable.  They  withstand  dry  heat,  but  are 
destroyed  in  watery  solutions  by  a  temperature  of  over 
70°  C.  They  are  injured  by  acids,  especially  the 
morganic  ones,  but  are  resistant  to  all  alkalies.  All 
fermentation  has  for  its  object  the  acquisition  by  the 
organism  of  a  store  of  energy.  This  is  accomplished 
in  either  of  the  ways  above  mentioned.  The  simplest 
and  commonest  example  of  decomposing  fermentation 
produced  by  an  enzyme  is  that  of  sugar  : 

C6H12O6        =        2C2H6O        +        2CO2 
Grape-sugar.  2  Alcohol.          2  Carbon  dioxide. 

or, 

C6H1206        =       2C3H603 
Grape-sugar.  2  Milk-sugar. 

or, 

CVH12O6        =        3C2H4O2 
Grape-sugar.  3  Acetic  acid. 

Bacteria  which  develop  in  the  absence  of  oxygen  are 
especially  in  need  of  this  source  of  oxygen.  Anaerobic 
bacteria,  for  this  reason,  have  the  power  of  decomposing 
sugar,  while  many  facultative  anaerobes  are  only  capable 
of  producing  fermentation  when  oxygen  is  excluded. 

Opposite  to  this,  and  far  less  common,  is  oxidizing 
fermentation,  as  in  the  production  of  acetic  acid  from 
alcohol.  Here  the  energy  is  acquired  not  by  the  de- 
composition but  by  the  oxidation  of  the  alcohol. 

The  proteolytic  or  peptonizing  ferments  which  are 
somewhat  analogous  to  pepsin  and  trypsin — being  capa- 
ble of  changing  albuminous  bodies  into  soluble  and 
diffusible  substances — are  very  widely  distributed. 
The  liquefaction  of  gelatin,  which  is  chemically  allied 


VITAL  PHENOMENA  OF  BACTERIA,  65 

to  albumin,  is  due  to  the  presence  of  a  proteolytic  fer- 
ment or  trypsin.  It  is  not  pepsin,  as  pepsin  acts  only 
in  the  presence  of  acid,  and  gelatin  is  liquefied  with  an 
alkaline  reaction  only.  The  production  of  proteolytic 
ferments  by  different  cultures  of  the  same  varieties  of 
bacteria  varies  considerably,  far  more  than  is  generally 
supposed.  Even  among  the  freely  liquefying  bacteria, 
such  as  the  cholera  spirillum  and  the  staphylococcus, 
poorly  liquefying  varieties  have  been  repeatedly  found. 
These  observations  have  detracted  considerably  from 
the  value  in  cultures  of  the  property  of  liquefying 
gelatin  as  a  positive  diagnostic  characteristic.  Most 
conditions  which  are  unfavorable  to  the  growth  of  bac- 
teria seem  to  interfere  also  with  their  liquefying  power. 

Certain  bitter-tasting  products  of  decomposition  are 
formed  by  liquefying  bacteria  in  media  containing  albu- 
min, as,  for  example,  in  milk. 

Diastatic  ferments  convert  starch  into  sugar.  That 
these  are  produced  by  bacteria  is  shown  by  mixing 
starch  paste  containing  1  per  cent,  thymol  with  cultures 
to  which  1  to  2  per  cent,  thymol  has  been  added,  and 
keeping  the  mixture  for  six  to  eight  hours  in  the  incu- 
bating oven;  then,  on  the  addition  of  Fehling's  solution 
and  heating,  the  reaction  for  sugar  appears — the  red- 
dish-yellow precipitate  due  to  the  reduction  of  the 
copper.  Bacteria  may  be  directly  tested  for  sugar  also 
by  boiling  potato-broth  cultures  and  using  the  extract. 

Inverting  ferments — that  is,  those  which  convert  cane- 
sugar  into  grape-sugar — are  of  very  frequent  occurrence. 
Bacterial  invertin  withstands  a  temperature  of  100°  C. 
for  more  than  an  hour,  and  is  produced  in  culture  media 
free  from  albumin. 

Rennet  ferments — substances  having  the  power  of 

5 


66  BACTERIOLOGY. 

coagulating  milk  with  neutral  reaction,  independent  of 
acids — are  found  not  infrequently  among  bacteria.  The 
B.  prodigiosus,  for  instance,  in  from  one  to  two  days 
coagulates  to  a  solid  mass  milk  which  has  been  steril- 
ized at  55°  to  60°  C.  These  ferments  have  not  been 
thoroughly  investigated:  they  are  probably  present, 
however,  in  all  species  of  bacteria  which  coagulate  milk 
with  the  production  of  acid. 

Fermentation  yields  products  that  are  poisonous  to 
the  ferment;  hence  fermentation  ceases  when  the  nu- 
triment is  exhausted  or  the  fermentation  is  in  excess. 
Different  kinds  of  fermentation  obtain  specific  names, 
according  to  product.  Thus  acetic,  yielding  acetic 
acid;  alcoholic  or  vinous,  yielding  alcohol;  ammoniacal, 
yielding  ammonia;  amylic,  yielding  amylic  alcohol; 
benzole,  yielding  benzoic  acid;  butyric,  yielding  butyric 
acid;  lactic,  yielding  lactic  acid;  and  viscous,  yielding 
a  gummy  mass. 

Pigment  Production.  Pigments  have  been  little  chem- 
ically studied,  but  the  recent  investigations  of  Klein 
and  Migula,  Thumm  and  Schneider,  and  others  throw 
some  light  on  the  subject.  They  have  no  known  im- 
portance in  connection  with  disease,  but  are  of  interest 
and  have  value  in  identifying  bacteria. 

RED  AND  YELLOW  PIGMENTS.  Of  the  twenty-seven 
red  and  yellow  bacteria  studied  by  Schneider,  almost 
all  produce  pigments  soluble  in  alcohol  and  insoluble 
in  water.  The  larger  majority  of  these  possess  in  com- 
mon the  property  of  being  colored  blue-green  by  sul- 
phuric acid  and  red  or  orange  by  a  solution  of  potash. 
Though  varying  considerably  in  their  chemical  compo- 
sition and  in  their  spectra,  they  may  be  classified,  for 
the  most  part,  among  that  large  group  of  pigments 


VITAL  PHENOMENA  OF  BACTERIA.  67 

common  to  both  the  animal  and  vegetable  kingdoms 
known  as  lipochromes,  and  to  which  belong  the  pig- 
ments of  fat,  yolk  of  egg,  the  carotin  of  carrots,  tur- 
nips, etc. 

VIOLET  PIGMENTS.  Certain  bacteria  produce  violet 
pigments,  also  insoluble  in  water  and  soluble  in  alcohol, 
but  insoluble  in  ether,  benzol,  and  chloroform.  These 
are  colored  yellow  when  treated  in  a  dry  state  with  sul- 
phuric acid  and  ernerald-green  with  potash  solution. 

BLUE  PIGMENTS  are  also  produced  by  the  so-called 
fluorescent  bacteria,  along  with  a  pigment  named  bac- 
terio-fluorescein.  In  cultures  the  fluorescence  is  at  first 
blue;  later,  as  the  cultures  become  alkaline,  it  is  green. 

Numerous  investigations  have  been  made  to  deter- 
mine the  cause  of  the  variation  in  the  chrornogenic 
function  of  bacteria.  All  conditions  which  are  unfav- 
orable to  the  growth  of  the  bacteria  decrease  the  pro- 
duction of  pigment,  as  cultivation  in  unsuitable  media 
or  at  too  low  or  too  high  a  temperature,  etc.  The  B. 
prodigiosus  produce  no  pigment  at  37°  0.,  and  when 
transplanted  at  this  temperature,  even  into  favorable 
media,  the  power  of  pigment  production  is  gradually 
lost. 

Otherwise  colorless  species  of  bacteria  sometimes  pro- 
duce pigments.  Thus  yellow  to  red  colonies  of  the 
pneutnococcus  have  been  observed,  and  colored  varie- 
ties of  the  streptococcus  pyogenes.  Occasionally  colored 
and  uncolored  colonies  of  the  same  species  of  bacteria 
may  be  seen  to  occur  side  by  side  in  one  plate  culture, 
as,  for  example,  the  staphylococcus  pyogenes. 

Alkaline  Products  and  the  Fermentation  of  Urea.  Aero- 
bic bacteria  sometimes  produce  alkaline  products  from 
albuminous  substances  in  culture  media  free  from  sugar. 


68  BACTERIOLOGY. 

Most  species  of  bacteria  produce  acids  in  the  presence 
of  sugar,  which  explains  the  fact  that  neutral  or  slightly 
alkaline  cultures  become  acid  at  first  from  the  small 
amount  of  sugar  contained  in  the  meat  used  for  making 
the  media.  When  the  sugar  is  used  up  the  reaction 
often  becomes  alkaline,  as  the  production  of  alkalies 
continues  after  the  acid  formation  has  ceased.  The 
substances  producing  the  alkalinity  in  cultures  are 
chiefly  ammonia,  amine,  and  the  ammonium  bases. 

The  conversion  of  urea  into  carbonate  of  ammonia 
affords  special  evidence  of  the  production  of  alkaline 
substances  by  bacteria: 

CO(NH2)2        -f        2H20  CO3(NH4)2 

Urea.  2  Water.  Ammonium  carbonate. 

Leube  has  isolated  several  organisms  from  putrefy- 
ing urine  which  separate  ammonia  from  urea.  The 
power  of  decomposing  urea,  however,  is  not  wide-spread 
among  bacteria.  Out  of  twenty-seven  organisms  studied 
by  Wariugton,  only  two  were  found  to  decompose  urea. 
Of  sixty  species  investigated  by  Lehmaun,  three  only 
developed  the  odor  of  ammonia  from  sterilized  human 
urine. 

Ptomains — Toxins.  But  beside  ammonium  carbonate, 
a  large  number  of  basic  crystalline  substances  have  been 
recognized,  especially  by  Brieger,  as  products  of  bacterial 
growth.  These  are  now  commonly  known  as  ptoma'im, 
or  putrefactive  alkaloids  (from  XTM/UX,  putrefaction). 

Nencki,  and  then  later  Brieger,  Vaughan  and  others, 
succeeded  in  preparing  organic  bases  of  a  definite  chem- 
ical composition  out  of  decomposing  fluids — meat,  fish, 
old  cheese,  and  milk  undergoing  bacterial  decomposition 
— as  well  as  from  pure  bacterial  cultures.  Some  of  these 
were  found  to  exert  a  poisonous  effect,  and  for  a  long  time 


VITAL  PHENOMENA  OF  BACTERIA.  69 

were  looked  upon  as  the  specific  bacterial  poisons,  while 
others  were  harmless.  The  poisons  are  particularly  in- 
teresting, since  they  may  be  present  in  the  decomposing 
cadaver  (hence  the  name  ptomai'n),  and,  in  consequence, 
have  to  be  taken  into  consideration  in  questions  of  legal 
medicine.  They  may  be  formed  also  in  the  living 
human  body,  and,  if  not  made  harmless  by  oxidation, 
may  come  to  act  therein  as  self-poisons  or  leuconiai'ns. 
They  are  now  known  not  to  be  the  substances  to  which 
are  due  the  specific  poisonous  effects  of  bacteria  which 
are  designated  as  toxins,  and  have  entirely  different 
characteristics. 

Many  ptomaios  are  known  already  and  the  empirical 
formula  of  each  made  out,  and  among  them  are  some 
whose  exact  chemical  composition  is  established.  The 
first  of  these  bodies  to  be  separated  was  colloidin 
(C8HUN),  obtained  byNencki  from  putrefying  gelatin. 
Another,  trimethylamin  (C3H9N  =  (CH3)3N),  gives  an 
odor  like  herring-brine.  Especially  interesting  is  the 
substance  cadaverin,  which  was  separated  by  Brieger 
from  portions  of  decomposing  dead  bodies  and  from 
cholera  cultures,  by  reason  of  the  fact  that  Ladenburg 
prepared  it  synthetically  and  showed  it  to  be  penta- 
methylenediamin  [(NH2)2(CH2)6].  The  cholin  group 
is  particularly  interesting.  Cholin  itself  (C5H15NO2) 
arises  from  the  hydrolytic  breaking-up  of  lecithin,  the 
fatty  substance  found  in  brain  tissue  and  other  nervous 
tissue. 

By  the  oxidation  of  cholin  there  can  be  produced  the 
non-poisonous  betain  or  trirnethylglycocoll  occurring  in 
beet-juice,  and  the  highly  toxic  muscarin,  found  by 
Schmiedeberg  in  a  poisonous  toadstool  and  by  Brieger 
in  certain  decomposing  substances  : 


70  BACTERIOLOGY. 

C5H15N02     +     02  C5H13N03     +     H20. 

Cholin,  Betain. 

C5H15N02     +     O  C5H13N03 

Cholin.  Muscarin. 

The  ptomai'n  tyrotoxiu,  obtained  from  cheese  by 
Vaughan,  is  apparently  derived  from  butyric  acid. 

Pyocyanin  (C14H14N2O),  which  produces  the  color  of 
blue  or  blue-green  pus,  and  has  been  regarded  by  Led- 
derhose  as  related  to  the  coal-tar  products,  is  a  ptomainic 
pigment.  Similar  bodies  of  a  basic  nature  may  be  found 
in  the  intestinal  contents  as  the  products  of  bacterial 
decomposition.  Some  of  these  are  poisons  and  can  be 
absorbed  into  the  body,  where  they  play  the  role  of 
self-poisons,  or  leucomai'ns.  Some  believe  the  symp- 
toms designated  as  coma  and  tetany  may  be  ascribed  to 
the  absorption  of  substances  of  this  nature.  Since  the 
name  ptomai'n  was  given  to  the  poisonous  products  of 
bacterial  growth  before  these  products  were  chemically 
understood,  and  even  now,  when  the  name  is  restricted 
to  crystalline  bodies,  it  is  by  many  frequently  applied 
to  all  bacterial  poisons,  as  in  cases  of  poisoning  due  to 
decomposing  meat  or  sausage  or  to  cheese  or  milk. 
Instead  of  ptomai'ns  these  may  be  due  to  the  poisonous 
proteids  or  toxins.  Such  poisonous  proteid  bodies  are 
always  formed  in  the  beginning  of  decomposition  pro- 
cesses. Some  of  the  ptomai'ns  obtained  by  chemists 
are  due  not  to  putrefactive  changes  but  to  the  chemi- 
cal methods  used  to  obtain  them. 

The  isolation  of  these  substances  can  here  be  only 
briefly  referred  to.  According  to  Brieger's  method, 
which  is  the  one  now  generally  employed,  the  cultures 
having  a  slight  acid  reaction  (HC1)  are  boiled  down, 
filtered,  and  the  filtrate  concentrated  to  a  syrupy  con- 


VITAL  PHENOMENA  OF  BACTERIA.  7] 

sistency.  This  is  then  dissolved  in  96  per  cent,  alcohol, 
freed  from  albumin  and  other  contamination  by  an 
alcoholic  solution  of  lead  acetate,  the  lead  precipitated, 
the  filtrate  concentrated,  and  again  precipitated  by  an 
alcoholic  solution  of  mercuric  chloride,  which  forms  a 
double  mercury  compound  with  the  ptomai'n.  The 
alcohol  is  evaporated  by  heat,  the  mercury  separated 
by  sulphuretted  hydrogen,  and  a  double  compound 
formed  with  gold  and  platinum,  the  crystal lizability  of 
which  permits  of  its  purification;  or  the  crystalline  hydro- 
chloride  is  directly  obtained,  and  the  free  bases, which 
are  often  liquid,  separated  by  means  of  sodium  hydrate. 

Many  of  these  ptomai'ns,  like  most  vegetable  alka- 
loids when  they  are  are  set  free  by  sodium  or  potassium 
hydrate,  are  obtainable  by  agitation  with  ether  in  aque- 
ous solution;  but  Brieger's  method  is  preferable,  because 
many  substances  not  taken  up  by  ether  are  here  ex- 
tracted. 

Complex  Albuminoid  Poisons — Toxalbumins  or  Toxins. 
These  may  be  divided  into  two  classes: 

1.  BACTERIAL  PROTEINS  (Buchner).  By  these  are 
understood  poisonous  substances  of  a  proteid  nature 
produced  by  bacteria  which  are  not  affected  by  heat, 
which  are  capable  of  producing  fever  (pyogenic)  and 
causing  inflammation  (phlogogenic),  and  which  can  be 
obtained  by  boiling  for  several  hours  potato  cultures 
treated  with  an  0.5  per  cent,  solution  of  potassium 
hydrate  (about  50  volumes  of  potassium  hydrate  to  1 
volume  of  bacterial  substance).  From  the  clear,  fil- 
tered liquid  the  proteins  are  precipitated  by  weak  acid, 
carefully  added,  and  the  precipitate  washed  and  dried; 
before  use  they  can  be  dissolved  in  weak  soda  solution. 

The  best  known  protein  is  Koch's  old  tuberculin; 


72  BACTERIOLOGY. 

mallein  is  another.  According  to  Buchner  and  Romer, 
all  bacterial  proteins  are  very  similar  in  their  action, 
and  Mathes  maintains  that  deutero-alburuose,  which  is 
obtained  by  the  action  of  pepsin  on  albumin  and  has 
no  connection  with  bacteria,  has  an  effect  on  tubercular 
guinea-pigs  somewhat  similar  to  tuberculin. 

Toxins— Active  Proteids.  Tox ALBUMINS.  Fraenkel 
and  Brieger,  confirming  the  statements  of  previous  in- 
vestigators (Christmas,  Roux  and  Yersin,  and  Hankin), 
have  shown  that  amorphous  poisons  having  an  intense 
and  often  specific  toxic  action — that  is  an  effect  similar 
to  that  produced  by  infection  with  the  living  organism 
— may  be  precipitated  from  bouillon  cultures  by  agents 
precipitating  albumin.  They  therefore  called  these 
poisons  "toxalburnins,"  and  regarded  them  as  being 
analogous  to  the  toxalbumins  of  vegetable  origin,  like 
ricin  from  the  castor-oil  bean  (Ricinis  communis), 
and  abrin  from  the  jequirity  bean  (Abras  precatorius). 
The  majority  of  investigators,  however,  consider  these 
poisons  to  be  "labile"1  albuminous  substances,  which 
are  derived  from  the  bacterial  cells.  Some  have  assumed 
that  they  were  similar  to  snake  poisons  or  to  the 
enzymes.  Like  these  substances,  they  are  very  sensi- 
tive to  the  action  of  heat,  chemical  agents,  light,  etc. 

Toxalbumins  are  obtained  as  crude  substances  by  the 
precipitation  of  the  products  of  fully  developed  cultures 
in  bouillon  under  a  vacuum,  with  absolute  alcohol  or 
ammonium  sulphate,  after  the  culture  fluid  has  been 
freed  from  living  germs  by  its  passage  through  a  por- 
celain filter.  When  ammonium  sulphate  is  used  the 
precipitate  is  freed  from  this  reagent  by  dialysis 

1  So-called  "labile"  substances  are  such  as  are  proue  to  undergo  chemical 
changes  or  alterations  of  atomic  structure ;  unstable. 


VITAL  PHENOMENA  OF  BACTERIA.  73 

through  parchment  against  running  water,  and  after 
concentration  the  substances  are  again  precipitated  by 
absolute  alcohol.  Recently  it  has  been  found  that  zinc 
chloride  separates  these  bodies  quantitatively,  and  that 
the  toxins  may  be  obtained  from  this  precipitate  by 
means  of  sodium  phosphate  (Brieger  and  Boer). 

All  along,  however,  some  doubt  has  been  expressed 
as  to  whether  these  so-called  toxalbumins  were  really 
only  obtainable  by  precipitation  from  albumin  or 
whether  they  had  anything  to  do  with  albumin  at 
all.  With  regard  to  tetanus  poison,  Brieger  and 
Cohn  have  now  succeeded  in  obtaining  what  they 
consider  an  almost  pure  toxin  from  the  crude  poison  by 
means  of  acetate  of  lead  and  ammonia.  This  substance 
gives  a  slight  violet  color  with  copper  sulphate  and 
soda  solution,  but  otherwise  no  albumin  reaction;  it 
contains  neither  phosphorus  nor  sulphur,  and  is  appar- 
ently not  an  albuminous  substance.  The  statement 
previously  made  by  Uschinsky  that  he  had  obtained 
albuminoid  tetanus  and  diphtheria  poisons  in  culture 
media  devoid  of  albumin  could  not,  heretofore,  be  con- 
firmed, owing  to  the  difficulty  experienced  by  most 
investigators  in  getting  a  sufficient  growth  of  these 
organisms  on  such  media.  Brieger  and  Cohu  have 
found  that  cholera  spirilla  produce  a  non-albuminous 
poison  in  Uschinsky' s  culture  media  (free  from  albu- 
min); and  now  diphtheria  toxin  has  been  recognized  to 
be  non-albuminous  (Brieger  and  Boer).  It  is  becoming 
more  and  more  customary  to  call  proteid  bacterial  poisons 
simply  toxins ,  irrespective  of  their  composition,  and  to 
ignore  the  existence  of  the  above- described  crystallizable 
toxins  of  simple  constitution., 

With  regard  to  the  other  properties  of  these  toxins, 


74  BACTERIOLOGY. 

taking  tetanus  toxin  as  an  example,  it  may  be  said  that 
in  aqueous  solution  it  is  not  coagulated  by  heat,  but  is 
in  time  deprived  of  its  poisonous  qualities.  The  addi- 
tion of  small  quantities  of  acids  or  alkalies  to  the  solu- 
tion, and  the  continued  passage  through  it  of  carbon 
dioxide  or  sulphuretted  hydrogen,  distinctly  reduce  its 
toxicity.  When  exposed  to  light  and  air,  either  in  a 
dry  state  or  in  solution,  the  toxin  deteriorates  rather 
rapidly.  It  withstands  a  temperature  of  70°  C.  for 
some  time  without  being  wholly  destroyed  ;  higher 
temperatures  decompose  it  rapidly.  When  protected 
from  the  light  and  air  it  is  slowly  converted  into  an  in- 
active substance;  it  is  better  preserved  under  absolute 
alcohol,  pure  ether,  and  the  like.  The  toxicity  of  the 
purest  tetanus  toxin  now  obtainable  is  almost  incred- 
ible:  0.00005  milligramme  of  it  kills  a  mouse  of  15 
grammes;  a  man  of  150  pounds  weight,  if  he  were  equally 
susceptible,  would  be  killed  with  0.23  milligrammes. 
It  requires  30  to  100  milligrammes  of  strychnine  to 
kill  a  man  under  ordinary  circumstances.  The  most 
virulent  diphtheria  bacilli  produce  a  specific  poison 
which  does  not  fall  far  behind  that  of  tetanus  in  power. 
Sulphuretted  Hydrogen.  Sulphuretted  hydrogen  is  a 
very  common  bacterial  product.  Its  presence  is  deter- 
mined by  pasting  a  piece  of  paper  moistened  with  lead 
acetate  inside  the  neck  of  the  flask  containing  the  cul- 
ture, closing  the  mouth  with  a  cotton-wool  stopper,  and 
over  this  again  an  India-rubber  cap  (black  rubber  free 
from  sulphur).  The  paper  is  colored  at  first  brownish 
and  later  black;  repeated  observation  is  necessary,  as 
the  color  sometimes  disappears  toward  the  end  of  the 
reaction.  Apparently  negative  results  should  not  be 
rashly  accepted  as  conclusive. 


VITAL  PHENOMENA  OF  BACTERIA. 

Sulphuretted  hydrogen  may  be  formed: 

1.  From  albuminous  substances.     This  power,  ac- 
cording to  Petri  and  Maassen,  of  forming  sulphuretted 
hydrogen,  particularly  in  liquid  culture  media  contain- 
ing much  peptone  (5  to  10  per  cent.)  and  no  sugar,  is 
possessed,  though  in  different  degree,  by  all  bacteria 
examined  by  them;  only  a  few  bacteria  form  H2S  in 
bouillon  in  the  absence  of  peptone,  while  about  50  per 
cent,  in  media  containing  1  per  cent,  peptone. 

2.  From  powdered  sulphur.     All  bacteria  produce  in 
culture  media  to  which  pure  powdered  sulphur  is  added 
considerably   more    H2S    than  without   this   addition. 
Petri   and  Maassen    suggest   that   this   is  due  to  the 
nascent  hydrogen  produced  by  the  bacteria. 

3.  From  thiosulphates  and  sulphites.     Studied  par- 
ticularly in  yeast,  but  demonstrated  also  by  Petri  and 
Maassen  in  several  bacteria. 

The  presence  of  sugar  in  the  culture  does  not  affect 
the  production  of  H2S  by  bacteria,  but  saltpetre  reduces 
it,  nitrites  being  formed.  The  absence  of  oxygen  favors 
the  production  of  H2S.  Light  diminishes  the  develop- 
ment of  H2S  by  facultative  anaerobes,  sulphates  being  . 
formed  instead. 

Reduction  Processes.  All  bacteria,  as  we  have  seen, 
possess  the  property  of  converting  sulphur  into  sul- 
phuretted hydrogen,  for  which  purpose  is  required  the 
presence  of  nascent  hydrogen.  The  following  pro- 
cesses depend  also  in  part  upon  the  action  of  nascent 
hydrogen : 

1.  The  reduction  of  blue  litmus  pigments,  methylene- 
blue,  and  indigo  to  colorless  substances.  The  superficial 
layer  of  cultures  in  contact  with  the  air  shows  often  no 
reduction,  only  the  deeper  layers  being  affected.  By 


76  BACTERIOLOGY. 

agitation  with  access  of  air  the  colors  may  be  again  re- 
stored, but  at  the  same  time,  acid  being  formed,  the 
litmus  pigment  is  turned  red.  According  to  Colin,  the 
property  of  reducing  litmus  belongs  to  all  liquefying 
bacteria,  but  some  non-liquefying  species  also  possess  it. 

2.  The  reduction  of  nitrates  to  nitrites  and  am- 
monia. The  first  of  these  properties  seems  to  pertain 
to  a  great  many  bacteria — at  least  Petri  and  Maassen 
found  in  six  species,  grown  in  bouillon  containing  2.5 
to  5  per  cent,  peptone  and  0.5  per  cent,  nitrate,  that 
almost  all  produced  nitrite  abundantly;  once  only  was 
ammonia  observed.  In  a  number  of  bacteria  studied 
by  Rubner  only  one  failed  to  produce  nitrite.  The 
test  for  nitrites  is  made  as  follows  :  Two  bouillon  tubes 
containing  nitrates  are  inoculated,  and,  along  with  two 
unirioculated  tubes,  are  allowed  to  remain  in  the  incu- 
bator for  several  days;  then  to  the  cultures  and  control 
test  is  added  a  small  quantity  of  colorless  iodide  of 
starch  solution  (thin  starch-paste  containing  0.5  per 
cent,  potassium  iodide)  and  a  few  drops  of  pure  sul- 
phuric acid.  The  control  tubes  remain  colorless  or 
become  gradually  slightly  blue,  while  if  nitrites  are 
present  a  dark  blue  or  brown-red  coloration  is  produced, 

The  demonstration  of  ammonia  is  made  by  the  addi- 
tion of  Nessler's  reagent  to  culture  media  free  from 
sugar.  In  bouillon,  if  ammonia  be  present,  Nessler's 
reagent  is  almost  immediately  reduced  to  black  mer- 
curous  oxide.  A  strip  of  paper  saturated  with  the 
reagent  can  also  be  suspended  over  the  bouillon  tube, 
or  this  can  be  distilled  with  the  addition  of  magnesium 
oxide  and  the  distillate  treated  with  Nessler's  reagent. 
A  yellow  to  red  coloration  indicates  the  presence  of 
ammonia.  Controls  are  necessary. 


VITAL  PHENOMENA  OF  BACTERIA.  77 

Aromatic  Products  of  Decomposition.  Many  bacteria 
produce  aromatic  substances  as  the  result  of  their 
growth.  The  best  known  of  these  are  indol,  skatol, 
phenol,  and  ty rosin.  Systematic  investigations  have 
only  been  made  with  regard  to  the  occurrence  of  indol 
and  phenol. 

Test  for  Indol.  To  a  bouillon  culture,  which  should, 
if  possible,  be  not  under  eight  days  old  and  free  from 
sugar,  is  added  half  its  volume  of  10  per  cent,  sulphuric 
acid.  If  in  heating  to  about  80°  C.  a  pink  or  bluish- 
pink  coloration  is  immediately  produced  it  indicates  the 
presence  of  both  indol  and  nitrites,  the  above-described 
nitroso-indol  reaction  requiring  the  presence  of  both  of 
these  substances  for  its  successful  operation.  This  is 
the  so-called  "  cholera-red  reaction/'  but  it  may  be 
applied  to  many  other  spirilla  beside  cholera.  As  a 
rule,  however,  the  addition  of  sulphuric  acid  alone  is 
not  sufficient,  and  a  little  nitrite  must  be  added;  this 
may  be  done  later,  the  culture  being  first  warmed  with- 
out ^nitrite,  when  if  there  is  no  reaction  or  a  doubtful 
one,  1  to  2  c.c.  of  a  0.5  per  cent,  solution  of  sodium 
nitrite  is  added  until  the  maximum  reaction  is  obtained. 
The  addition  of  strong  solutions  of  nitrite  colors  the 
acid  liquid  brownish-yellow  and  ruins  the  test. 

Out  of  sixty  species  examined  by  Lehmann,  twenty- 
three  gave  the  indol  reaction.  Levandoosky  states  that 
the  color  group  in  general,  glanders,  diphtheria,  proteus 
vulgaris,  and  most  of  the  spirilla,  are  indol  producers; 
with  the  exception  of  the  spirilla,  these  bacteria  also 
produce  phenol. 

Decomposition  of  Fats.  Pure  melted  butter  is  not  a 
suitable  culture  medium  for  bacteria.  The  rancidity 
of  butter  is  brought  about  (1)  ak  the  result  of  a  purely 


\ 


78  BACTERIOLOGY. 

chemical  decomposition  of  the  butter  by  the  oxygen  of 
the  air  under  the  influence  of  sunlight  and  (2)  through 
fermentation  by  the  lactic  acid  of  the  milk-sugar  left 
in  the  butter.  Fats  are,  however,  attacked  by  bacteria 
when  mixed  with  gelatin  and  used  as  culture  media, 
with  the  consequent  production  of  acid. 

Putrefaction.  By  putrefaction  is  understood  in  com- 
mon parlance  every  kind  of  decomposition  due  to  bac- 
teria which  results  in  the  production  of  malodorous 
substances.  Scientifically  considered,  putrefaction  de- 
pends upon  the  decomposition  of  complex  organic 
compounds,  albuminous  substances,  and  the  like  (glue, 
albuminoid  bodies),  which  are  frequently  first  pepton- 
ized  and  then  further  decomposed.  Typical  putrefac- 
tion occurs  only  when  oxygen  is  absent  or  scanty;  the 
free  passage  of  air  through  a  culture  of  putrefactive 
bacteria — an  event  which  does  not  take  place  in  natural 
putrefaction — very  much  modifies  the  process :  first, 
biologically,  as  the  anaerobic  bacteria  are  inhibited, 
and  then  by  the  action  of  the  oxygen  on  the  products 
or  by-products  of  the  aerobic  and  facultative  anaerobic 
bacteria. 

As  putrefactive  products  we  have  peptone,  ammonia 
and  amines,  leucin,  tyrosin,  and  other  amido  substances. 
Oxyfatty  acids,  indol,  skatol,  phenol,  and,  finally,  sul- 
phuretted hydrogen,  mercaptan,  carbonic  acid,  hydro- 
gen, and,  possibly,  marsh-gas  (H4C). 

According  to  recent  observations,  nitrification  is 
produced  by  a  small,  special  group  of  bacteria,  culti- 
vated with  difficulty,  which  do  not  grow  on  our  usual 
culture  media.  From  the  investigations  of  Winograd- 
sky  it  would  appear  that  there  are  two  common  micro- 
organisms present  in  the  soil,  one  of  which  converts 


VITAL  PHENOMENA  OF  BACTERIA.  79 

ammonia  into  nitrites  and  the  other  converts  nitrites 
into  nitrates. 

Conversion  of  Nitrous  and  Nitric  Acids  into  Free  Nitrogen. 
This  process  is  performed  by  a  number  of  bacteria.  The 
special  nitrate- ferment  ing  bacteria,  however,  were  first 
accurately  described  by  Barri  and  Stutzer.  In  their 
exhaustive  investigation  they  first  isolated  from  horse- 
manure  two  bacteria,  neither  of  which  was  alone  capable 
of  producing  nitrogen  from  nitrates,  but  which  together 
in  the  presence  of  oxygen,  but  never  without  it  entirely, 
decomposed  nitrates  energetically.  Later  a  second 
denificating  bacillus  was  found,  B.  denitrificans  II., 
which  by  itself  was  able  to  produce  nitrogen  from 
nitrates. 

The  practical  importance  of  these  organisms  is  that 
by  their  action  large  quantities  of  nitrates  in  the  soil, 
and  especially  in  manure,  may  become  lost  as  plant- 
food  by  being  converted  into  nitrogen. 

Nitrogen  Combination.  The  bacillus  radicicola  of 
Beyerinck,  which  was  isolated  by  him,  has  the  power  of 
assimilating  nitrogen  from  the  air.  This  bacillus  is  found 
in  the  small  root-nodules  of  various  leguminous  plants 
(pease,  clover,  etc.),  and  can  be  obtained  from  these  in 
cultures.  Different  varieties  exist  in  different  kinds 
of  legumes,  each  kind  of  legume  apparently  having  a 
special  variety  of  bacteria  adapted  to  it,  and  not  every 
variety  is  capable  of  producing  nodules  in  all  legumes. 
There  are  certain  "  neutral  "  varieties,  however,  existing 
free  in  the  soil  and  not  adapted  to  any  special  legume, 
and  these  seem  to  be  able  to  form  nodules  in  different 
legumes. 

By  the  aid  of  these  root-bacteria,  which  gain  entrance 
to  the  roots  and  there  produce  this  nodular  formation, 


80  BACTERIOLOGY. 

the  leguminous  plants  are  enabled  to  assimilate  nitrogen 
from  the  atmosphere,  thus  yielding  harvests  of  grain, 
etc.,  which  are  highly  nitrogenous,  upon  soils  which  are 
naturally  poor  in  nitrogen.  This  explains  the  reason 
why  poor,  sandy  soils  become  gradually  fruitful  when 
pease,  lupine  and  other  varieties  of  legumes  are  grown 
upon  them  and  then  turned  under  with  the  plough.  It 
is  not  known  exactly  how  this  assimilation  of  nitrogen 
occurs,  but  it  is  assumed  that  the  zoogloea-li ke  bacteria, 
called  bacteroids,  constantly  observed  in  the  nodules, 
either  alone  or  in  a  special  degree,  possess  the  property 
of  assimilating  and.  combining  nitrogen.  It  seems, 
moreover,  to  have  been  recently  established  that,  in- 
dependently of  the  assistance  of  the  legumes,  certain 
nodule-bacteria  exist  free  in  the  soil,  which  accumulate 
nitrogen  by  absorbing  it  from  the  air  (Stutzer). 

Formation  of  Acids  from  Carbohydrates.  Free  acids 
are  formed  by  many  bacteria  in  culture  media  contain- 
ing sugar;  the  production  of  acid  in  ordinary  bouillon 
takes  place  on  account  of  the  presence  of  grape-sugar, 
which  is  usually  derived  in  small  quantities  from  the 
meat.1  According  to  Theobald  Smith,  all  anaerobic  or 
facultative  anaerobic  bacteria  form  acids  from  sugar; 
the  strict  aerobic  species  do  not,  or  so  very  slowly 
that  the  acid  is  concealed  by  the  almost  simultaneous 
production  of  alkali.  The  formation  of  acid  occurs 
sometimes  with  and  sometimes  without  the  production 
of  gas.  Excessive  acid  production  may  cause  the  death 
of  the  bacteria  from  the  increase  in  acidity  of  the 
culture  media. 


1  According  to  Theobald  Smith,  75  per  cent,  of  the  beef  ordinarily  bought 
in  the  markets  contains  appreciable  quantities  of  sugar  (up  to  0.3  per  cent.). 


VITAL  PHENOMENA  OF  BACTERIA.  81 

If  after  the  sugar  is  consumed  not  enough  acid  has 
been  formed  to  kill  the  bacteria,  a  similar  change  in 
reaction  now  takes  place  to  that  in  ordinary  culture 
media  in  the  absence  of  sugar — viz.,  the  acid  is  neu- 
tralized gradually,  and  in  the  end  the  reaction  becomes 
alkaline. 

Among  the  acids  produced  the  most  important  is 
lactic  acid;  also  traces  of  formic  acid,  acetic  acid,  pro- 
prionic  acid,  and  butyric  acid,  and  not  infrequently 
some  ethyl-alcohol  and  aldehyde  or  ac'etone  are  formed. 
Occasionally  no  lactic  acid  is  present,  and  only  the  other 
acids  are  formed. 

Various  bacteria,  as  yet  incompletely 'studied,  possess 
the  property  of  producing  butyric  acid  and  butyl-alcohol 
from  carbohydrates. 

Some  bacteria  also  seem  to  have  the  power  of  decom- 
posing cellulose,  found  in  the  stomach  and  intestinal 
contents  of  herbivorous  animals  and  in  marshy  soils, 
with  the  production  of  marsh-gas. 

Formation  of  Gas  from  Carbohydrates  and  Other  Ferment- 
able Substances  of  the  Fatty  Series.  The  only  gas  pro- 
duced in  visible  quantity  in  sugar-free  culture  media  is 
nitrogen.  If  sugar  is  vigorously  decomposed  by  bac- 
teria, as  long  as  pure  lactic  acid  or  acetic  acid  is  pro- 
duced there  may  be  no  development  of  gas,  as,  for 
instance,  with  the  B.  typhosus  on  grape-sugar;  but 
frequently  there  is  much  gas  developed,  especially  in 
the  absence  of  air.  About  one-third  of  the  acid-pro- 
ducing species  also  develop  gas  abundantly,  this  con- 
sisting chiefly  of  CO2,  which,  according  to  Smith,  is 
always  mixed  with  H.  Marsh-gas  is  seldom  formed 
by  bacteria,  with  the  exception  of  those  decomposing 
cellulose. 

6 


82 


BACTERIOLOGY. 


In  order  to  test  the  production  of  gas,  a  culture 
medium  composed  of  glucose-agar,  containing  about 
1  per  cent,  grape  sugar,  may  be  used.  At  the  end  of 
eight  to  twelve  hours  in  the  incubator  (or  twenty-four 
hours7  room-temperature)  the  agar  will  be  seen  to  be 
full  of  gas-bubbles  or  broken  up  into  holes  and  fissures. 

For  the  determination  of  the  quantity  and  kind  of 
gas  produced  by  a  given  micro-organism  the  fermenta- 
tion tube  recommended  by  Theobald  Smith  is  the  best. 
This  is  a  bent  tube,  constricted  greatly  at  its  lowest 
portion  (Eichorn's),  supported  upon  a  glass  base,  as 
shown  in  Fig.  14.  The  graduation  shown  in  the 

FIG.  14. 


Fermentation  tube  left  side,  ordinary  tube  on  right  side. 

upright  arm  is  not  essential  for  ordinary  laboratory 
work.  The  tube  is  filled  with  a  culture  media  consist- 
ing of  1  per  cent,  glucose,  peptone  bouillon  (without 
air-bubbles),  and  sterilized  in  the  steam  sterilizer.  It 
is  then  inoculated  with  a  loopful  of  a  culture  of  the 
organism  in  question,  and  observations  taken: 

1.   If  there  is  a  turbidity  produced  in  the  open  bulb 
it  indicates  the  presence  of  an  aerobic  species;  if  this 


VITAL  PHENOMENA  OF  BACTERIA.  83 

clouding  occurs  only  in  the  closed  arm,  while  the  open 
bulb  remains  clear,  it  is  an  anaerobic  species. 

2.  The   quantity  of  gas  produced   daily  should  be 
marked  on  the  upright  arm;  if  the  tube  is  graduated  a 
note  of  it  is  taken  and  the  percentage  calculated  on  the 
fourth  to  the  sixth  day  after  gas-production  has  ceased. 

3.  A  rough  analysis  of   the  gas  produced  may  be 
made  as  follows  :  Having  signified  by  a  mark  on  the 
tube  the  quantity  of  gas  produced,  the  open  bulb  is 
completely  filled  with  a  10  per  cent,  solution  of  soda, 
the  mouth  tightly  closed  with  the  thumb,  and  the  mix- 
ture thoroughly  shaken.    After  a  minute  or  two  all  the 
gas  is  allowed  to  rise  to  the  top  of  the  closed  arm  by 
inclining  and  turning  the  tube,  and  then,  removing  the 
thumb,  the  new  volume  of  gas  formed  is  permitted  to 
escape.     That  which  passes  off  is  carbonic  acid  gas; 
the  remainder  is  nitrogen,  hydrogen,  and  marsh-gas. 

For  the  quantitative  determination  of  these  gases 
Hein  pel's  gas  pipettes  may  be  used.  The  principle  of 
the  method  is  this  :  The  hydrogen,  mixed  with  oxygen 
and  passed  over  red-hot  palladium  asbestos,  becomes 
water,  and  thus  disappears;  the  carburetted  hydrogen 
is  converted  into  carbon-dioxide  and  is  estimated  as 
such;  the  remainder  is  nitrogen. 

Formation  of  Acids  from  Alcohol  and  Other  Organic  Acids. 
It  has  long  been  known  that  the  bacterium  acetiand  other 
allied  bacteria  convert  dilute  solutions  of  ethyl-alcohol, 
under  the  influence  of  oxidation,  into  acetic  acid: 
CH3    +    02    +    CH3    +    H20. 

CH2OH  COOH. 

The  higher  alcohols — glycerin,  dulcit,  mannit,  etc. 
— are  also  converted  into  acids — glycerin,  indeed,  as 
commonly  as  sugar. 


84  BACTERIOLOGY. 

Finally,  numerous  results  have  been  obtained  from 
the  conversion  of  the  fatty  acids  and  their  salts  into 
other  fatty  acids  by  bacteria.  As  a  rule,  the  lime-salts 
of  lactic,  malic,  tartaric,  and  citric  acids  have  been  em- 
ployed, these  being  converted  in  various  acids  by  the 
action  of  bacteria,  such  as  butyric,  priprionic,  valeri- 
anic,  and  acetic  acids;  also  succinic  acid,  ethyl-alcohol, 
and,  more  rarely,  formic  acid,  have  been  produced. 
Among  the  gases  formed  were  chiefly  CO2  and  H. 

Thus  Pasteur  found  that  anaerobic  bacteria  convert 
lactate  of  lime  into  butyric  acid. 


CHAPTER  IV. 

THE   RELATION    OF    BACTERIA   TO   DISEASE. 

IN  the  preceding  chapter  our  consideration  has  been 
given  largely  to  the  chemical  effects  of  bacteria  on  dead 
organic  substances.  Here  we  have  to  consider  the 
growth  of  bacteria  in  living  bodies  and  the  results  of 
such  development.  While  it  is  true  that  there  is  a 
great  difference  between  living  and  dead  matter,  and 
that,  therefore,  the  living  animal  cannot  be  considered 
as  merely  a  quantity  of  organic  and  inorganic  material, 
to  be  used  for  food  for  bacterial  growth,  still  the  fact 
that  bacteria  do  increase  in  the  living  body  shows 
that  its  tissues  are  under  certain  conditions  a  suitable 
nutrient  soil  for  their  growth.  In  a  sense,  therefore, 
we  are  warranted  to  consider  the  living  body  as  we  do 
any  other  medium  for  bacterial  growth,  remembering, 
however,  that  beside  the  chemical  nature,  temperature, 
etc.,  of  its  tissues,  micro-organisms  have  also  to  reckon 
with  the  mysterious  influence  of  life  with  which  all 
parts  of  the  body  are  endowed.  In  the  production  of 
disease  by  micro-organisms  there  are  two  main  factors 
involved — viz.,  the  power  to  elaborate  poison  and  the 
ability  to  multiply.  No  known  variety  of  bacterial  cell 
has  as  a  single  organism  the  ability  to  produce  enough 
poison  to  do  appreciable  injury  in  the  body,  nor  is  there 
any  variety  which  if  it  multiplied  in  the  body  to  the 
full  extent  to  which  it  is  capable  under  favorable  con- 


86  BACTERIOLOGY. 

ditions  will  not  produce  disease.  As  already  men- 
tioned, bacteria  even  under  similar  conditions  differ 
enormously  in  the  amount  of  poison  which  each  organ- 
ism produces  and  in  their  ability  after  gaining  entrance 
to  multiply  in  the  body. 

To  understands  all  the  production  of  disease  through 
bacteria  we  must  recognize  that  both  the  body  invaded 
and  the  bacteria  which  invade  are  living  organisms. 
They  are  in  bulk,  wide  apart,  but  both  have  life.  Just 
as  there  are  different  races  and  species  of  animals,  there 
are  different  races  and  species  among  bacteria,  and  just 
as  the  descendants  of  one  animal  species  under  changing 
conditions  gradually  become  diverse,  so  do  the  descend- 
ants of  one  bacterial  species.  Considering  these  facts, 
we  can  readily  understand  how  all  of  bacteria  do  not 
grow  equally  well  in  every  variety  of  animal,  nor  even 
find  the  body  of  the  same  animal  always  equally  suit- 
able. This  is  all  the  more  apparent  when  we  consider 
that  the  study  of  bacteria  in  the  more  simple  and 
known  conditions  of  artificial  culture  media  has  already 
shown  us  how  extremely  sensitive  many  bacteria  are  to 
slight  chemical,  thermal,  and  other  changes. 

Thus  if  we  take  specimens  of  diphtheria  bacilli  from 
three  different  cases  of  diphtheria,  we  find  that  on  grow- 
ing them  for  several  days  in  suitable  bouillon  one  will 
have  produced  poison  in  the  culture  fluid  to  such  a 
degree  that  one  drop  suffices  to  kill  a  large  guinea- 
pig;  the  second,  grown  in  a  similar  manner,  will  kill 
another  animal  of  the  same  size  with  half  a  drop; 
while  the  third  will  kill  with  one-tenth  of  a  drop. 
In  other  words,  different  varieties  of  diphtheria  bacilli 
under  similar  conditions  have  different  toxin-producing 
powers. 


RELATION  OF  BACTERIA   TO  DISEASE.         87 

Let  us  now  cultivate  these  same  bacilli  in  bouillon, 
which  is  a  little  too  acid  or  a  little  too  alkaline  for  their 
maximum  development,  and  we  shall  find  that,  while 
all  of  them  will  grow,  only  the  one  which  produced 
the  most  toxin  under  favorable  conditions  will  continue 
to  develop  it,  while  the  others  will  fail  to  produce 
any  specific  poison.  This  shows  that  growth  of  bac- 
teria may  occur  in  the  body  and  yet  no  specific  poison 
be  produced,  and  that  of  the  same  species  of  bacteria 
some  varieties  are  capable  of  producing  toxin  under 
less  favorable  circumstances  than  others. 

Slight  variations  in  the  culture  media  are,  moreover, 
of  great  importance  in  aiding  or  inhibiting  the  growth 
of  bacteria.  Thus  the  diphtheria  bacillus  grown  in 
neutral  bouillon  containing  a  little  glucose  will  at  first 
thrive  luxuriantly;  but  as  the  result  of  its  growth  fer- 
mentation of  the  glucose  takes  place  and  acid  is  pro- 
duced, which  then  inhibits  the  further  development  of 
the  bacillus.  After  a  while,  however,  the  glucose  having 
been  entirely  destroyed,  acid  formation  ceases  while 
alkaline  products  continue  to  be  formed,  and  thus  render 
the  medium  neutral  or  slightly  alkaline  again,  and  now 
a  vigorous  growth  again  starts  up. 

The  cultivation  of  the  tetanus  bacillus  also  furnishes 
some  interesting  facts  which  illustrate  the  complicated 
ways  in  which  bacterial  growth  is  hindered  or  assisted. 
The  tetanus  bacillus,  when  placed  in  suitable  media, 
will  not  grow  except  in  the  absence  of  oxygen;  but 
place  it  under  the  same  conditions,  together  with  a 
bacillus  which  actively  assimilates  oxygen,  and  the  two 
in  association  will  grow  in  the  presence  of  air. 

The  tubercle  bacillus,  when  taken  direct  from  an 
animal,  will  only  grow  in  a  few  selected  media,  such  as 


88  BACTERIOLOGY. 

blood-serum  media,  but  later  on  it  may  be  transplanted 
to  agar,  and  still  later  to  bouillon.  After  the  bacilli 
have  become  accustomed  to  the  bouillon  they  grow  with 
great  luxuriance,  but  only  when  carefully  floated  on  the 
surface  of  the  liquid.  If  submerged  in  the  slightest 
degree  they  will  not  grow. 

Many  bacteria  which  demand  free  access  of  oxygen 
grow  only  in  the  superficial  portion  of  the  nutrient 
agar  jelly,  where  there  is  plenty  of  air. 

It  is  evident,  therefore,  that  for  each  variety  of  organ- 
ism there  are  special  conditions  requisite  for  growth, 
and  that  a  temperature,  degree  of  acidity,  supply  of 
oxygen,  immersion  in  fluid,  etc.,  suitable  for  one  may 
be  utterly  unsuitable  for  another;  that,  still  further, 
when  two  organisms  grow  together  one  may  so  alter 
some  of  these  conditions  as  to  render  unsuitable  ones 
suitable,  and  vice  versa.  Since,  therefore,  bacteria  vary 
greatly  as  to  the  amount  of  toxin  which  they  produce, 
and  their  ability  to  develop  under  different  conditions 
outside  of  the  body,  we  should  certainly  expect  even 
greater  variations  in  the  living  bodies  of  men  and  ani- 
mals where  not  only  in  different  individuals,  but  even 
in  the  same  individual  at  different  times,  there  is  a 
varying  suitableness  for  such  growth. 

Let  us  now  consider  some  of  the  facts  which  have 
been  observed  concerning  the  growth  of  bacteria  in  the 
body,  and  then  endeavor,  as  far  as  possible,  to  explain 
them. 

In  the  first  place,  there  are  some  bacteria  which  find 
it  impossible  to  grow  in  the  living  body.  This  is  true 
of  the  great  mass  of  bacteria  occurring  in  the  air,  water, 
and  soil.  These  bacteria  cannot,  therefore,  produce 
infectious  diseases.  Some  of  them,  however,  produce 


RELATION  OF  BACTERIA  TO  DISEASE.         gg 

poisons  in  foods,  etc.,  which  are  absorbable,  and  which 
when  taken  with  food  or  drink  can  produce  a  chemical 
intoxication.  That  they  are  really  deleterious  is  shown 
by  the  fact  that  if  a  sufficient  quantity  of  their  pure 
cultures  is  injected  into  the  tissues  suppuration  and  ab- 
scesses are  produced  by  the  toxic  substances  contained 
within  them. 

Closely  allied  to  the  bacteria  which  cannot  grow  at 
all  in  the  bodies  of  warm-blooded  animals  are  those 
which  are  able  to  grow  in  or  upon  certain  circum- 
scribed areas  only.  Thus  the  diphtheria  bacilli  grow 
upon  the  abraded  mucous  membranes  of  the  respiratory 
tract,  but  cannot  develop  in  the  blood  or  in  the  subcu- 
taneous tissues.  The  cholera  spirilla  develop  in  the  in- 
flamed intestinal  mucous  membrane,  but  cannot  grow 
in  the  respiratory  tract,  blood,  or  tissues.  The  tetanus 
bacilli  develop  in  wounds  of  the  subcutaneous  tissues, 
but  cannot  grow  on  the  body-surface  or  in  the  blood. 

Another  group  of  bacteria  find,  indeed,  certain  regions 
most  suitable  in  their  conditions  for  growth,  but  under 
circumstances  favorable  for  them  are  capable  of  more  ex- 
tensive growth.  Thus  the  typhoid  bacillus  grows  most 
luxuriantly  in  the  Peyer's  patches  and  mesenteric  glands, 
but  also  invades  the  blood,  spleen,  and  other  regions. 
The  tubercle  bacillus  often  remains  localized  in  the 
apex  of  a  lung  or  a  gland  for  years,  but  at  any  time 
may  invade  many  tissues  of  the  body.  The  gonococcus 
finds  the  mucous  membrane  of  the  genito-urinary  tract 
most  suitable  for  its  development,  but  also  frequently  is 
capable  of  growth  in  the  peritoneum  and  even  some- 
times in  the  general  circulation.  The  pneumococcus 
develops  most  readily  in  the  lungs,  but  also  invades  the 
connective  tissues,  serous  membranes,  and  the  blood. 


90  BACTERIOLOGY. 

Still  further  removed  from  the  saprophytic  bacteria 
are  those  which  grow  in  the  blood  and  most  living 
tissues  as  readily  as  in  the  most  suitable  artificial 
media.  Thus  a  streptococcus  which  has  passed 
through  a  number  of  animals  or  human  beings  will, 
when  introduced  into  the  circulation  or  the  tissues, 
develop  as  rapidly  and  generally  as  in  bouillon,  and 
produce  death  within  twenty-four  hours,  every  drop  of 
blood  being  crowded  with  bacteria. 

Finally,  there  are  bacteria  which,  in  so  far  as  we 
know,  find  the  bodies  of  human  beings  or  animals  the 
only  fit  soil  for  their  growth.  These  are  the  true 
parasites.  The  leprosy  bacillus  grows  only  in  man; 
neither  the  food  nor  the  conditions  suitable  for  the  de- 
velopment of  this  micro-organism  outside  of  the  body 
have  as  yet  been  discovered.  The  spirillum  of  relapsing 
fever  is  another  good  example  of  this  group. 

Following  rather  closely  the  schematic  separation  of 
bacteria  according  to  their  relation  to  disease  we  might 
classify  them  as  : 

1.  Strict  saprophytes,  or  bacteria  which  grow  readily 
in  suitable  dead  organic  material,  but  not  in  the  body 
under  ordinary  conditions. 

a.  Bacteria  which  in  their  growth  produce  no  sub- 
stances which  are  poisonous  to  the  body,  or  at  least  none 
capable  of  absorption. 

b.  Bacteria  which  produce  in  their  growth  in  dead 
organic  matter  sufficient  poisons  to   cause  sickness  if 
they  are  absorbed  into  the  animal  body. 

2.  Facultative  Saprophytes.    These  are  bacteria  which 
can  develop  either  as  parasites  or  saprophytes.     The 
different  varieties  vary  as   to   the   amount  of   poison 
which  they  produce.     Some  grow  luxuriantly  in  dead 


RELATION  OF  BACTERIA  TO  DISEASE.        91 

organic  material  under  very  diverse  conditions,  others 
only  under  specially  favorable  conditions.  In  the  body 
they  also  vary — some  grow  extensively  in  the  blood, 
while  others  are  limited  to  one  or  more  tissues,  some 
being  widely  disseminated  throughout  the  body,  while 
others  are  localized  in  or  upon  a  certain  portion  of  it. 

3.  Strict  parasites,  or  bacteria  which,  so  far  as  we 
know,  grow  only  in  the  living  animal  or  vegetable 
organism.  These  again  vary  in  the  amount  of  poison 
which  they  produce  and  in  the  local  or  general  infec- 
tion they  give  rise  to. 

Adaptation  of  Bacteria  to  the  Soil  upon  which  They  are 
Grown.  Those  bacteria  which  grow  both  in  living  and 
dead  substances  vary  from  time  to  time  as  to  their 
readiness  to  develop  in  either  the  one  or  the  other. 
As  a  general  rule,  bacteria  grown  in  any  one  medium 
become  more  and  more  accustomed  to  that  and  other 
media  more  or  less  analogous  to  it,  while,  on  the  other 
hand,  they  are  less  easily  cultivated  on  media  widely 
different  from  that  in  which  they  have  developed.  Thus 
we  have  a  culture  of  tubercle  bacilli,  which,  after  having 
grown  for  three  years  in  the  bodies  of  guinea-pigs,  will 
no  longer  develop  on  dead  organic  matter,  while  a 
bacillus  which  was  obtained  from  the  same  stock,  but 
grown  on  bouillon  for  three  years,  will  no  longer  de- 
velop in  the  animal  body.  From  the  same  stock, 
therefore,  two  varieties  have  developed,  the  one  being 
now  practically  a  saprophyte  and  the  other  a  parasite. 

The  Local  Effects  Produced  by  Bacteria  and  their  Prod- 
ucts. Nearly  all  the  forms  of  acute  inflammation  are 
seen  to  follow  the  development  of  bacteria.  Thus  in- 
flammation and  serous  exudation  into  the  subcutaneous 
tissues  follow  injections  of  the  pneumococcus  or  anthrax 


92  BACTERIOLOGY. 

bacillus.  The  development  of  the  streptococcus  or 
pneumococcus  in  the  endocardium  or  pleural  cavity  is 
followed  by  a  serous  exudation,  frequently  with  more  or 
less  fibrin  production.  The  formation  of  pus  results, 
more  especially  from  the  streptococcus,  pneumococcus, 
and  staphylococcus;  but  also  nearly  all  forms  of  bacteria, 
when  they  accumulate  in  one  locality,  may  produce 
purulent  inflammation.  The  colon,  typhoid,  and  influ- 
enza bacilli  frequently  cause  the  formation  of  abscesses. 

Catarrhal  inflammation,  with  or  without  pus,  follows 
the  absorption  of  the  products  of  many  bacteria,  such  as 
the  gonococcus,  pneumococcus,  streptococcus,  and  in- 
fluenza bacillus,  etc.  The  hemorrhagic  exudation  seen 
in  pneumonia  is  due  to  the  pneumococcus;  it  is  observed 
also  in  anthrax  and  other  infections.  Cell  necrosis  is 
produced  frequently  by  the  products  of  the  diphtheria 
and  of  the  typhoid  bacilli  and  by  those  of  other  bac- 
teria. Specific  proliferative  inflammation  follows  the 
localization  of  the  products  derived  from  the  tubercle 
bacillus  and  the  leprosy  bacillus. 

Not  only  can  one  species  of  bacteria  produce  several 
forms  of  inflammation,  but  the  same  organism  will  vary 
as  to  the  kind  or  kinds  of  inflammation  it  will  produce; 
this  depending,  first,  upon  its  own  characteristics  at  the 
time  as  to  virulence,  etc.,  and,  second,  upon  the  con- 
ditions in  the  infected  animal,  such  as  its  health  and 
power  of  resistance,  the  period  of  infection,  and  the 
circumstances  under  which  the  animal  remains.  Such 
variations,  therefore,  are  in  no  case  specific,  for  different 
poisons  will  produce  changes  which  appear  identical. 

The  Manner  in  which  Bacteria  Produce  Disease.  The 
actual  mechanical  presence  of  the  bacteria  is  only  of 
importance  when,  as  in  septicaemia  or  pyiiemia,  tliey  exist 


RELATION  OF  BACTERIA  TO  DISEASE.         93 

in  such  enormous  numbers  as  to  interfere  mechanically 
with  the  circulation  or  cause  minute  thrombi,  and  later 
emboli,  which  finally  produce  infarction  and  abscesses 
in  different  parts  of  the  body.  These  dangerous  effects 
are  chiefly  due,  first,  to  their  alteration  of  the  nutritive 
substances  in  the  body  into  others  which  are  valueless, 
and,  second,  to  their  production  of  substances  which 
are  more  or  less  directly  poisonous. 

A  moment's  consideration  of  the  different  changes 
which  take  place  in  the  tissues  after  the  injection  of 
fine  sterile  sand  and  of  an  equal  quantity  of  a  dead 
culture  of  the  tubercle  or  typhoid  bacillus  would  suf- 
fice to  convince  any  one  that  it  was  the  poison  produced 
by  the  bacillus,  and  not  its  mechanical  interference, 
which  caused  disease.  These  poisonous  products,  as 
already  described  in  the  previous  chapter,  can  be 
separated  from  the  culture  fluid  in  which  the  bac- 
teria have  grown  or  they  can  be  extracted  from  their 
bodies.  These  products  without  the  bacteria  them- 
selves injected  into  animals  cause  essentially  the  same 
lesions  as  are  produced  by  the  bacteria  when  they  de- 
velop in  the  animal  body.  When  the  body,  as  a  whole, 
is  invaded  by  bacteria  the  abstraction  from  the  body 
of  such  substances  as  they  consume  exerts  probably  a 
considerable  influence;  but  even  here  it  is  the  poisons 
elaborated  by  bacteria  from  the  body  substances  and 
given  up  to  the  blood  and  tissue  cells  which  are  of  most 
importance.  The  substances  contained  in  or  produced 
by  the  bacteria,  with  few  exceptions,  attract  the  leuco- 
cytes, and  when  great  masses  of  bacteria  die  suppura- 
tion usually  follows. 

The  General  Symptoms  Caused  by  Bacterial  Poisons 
Absorbed  into  the  Circulation.  Fever  is  produced  under 


94  BACTERIOLOGY. 

favorable  conditions  by  all  bacterial  poisons.  The  first 
requisite  is  that  sufficient  poison  be  absorbed;  but,  on  the 
other  hand,  it  must  not  be  absorbed  with  such  rapidity  as 
to  overwhelm  the  injected  animal,  for  a  moderate  dose 
may  raise  the  temperature,  while  a  very  large  dose 
lowers  it,  as  occurs  sometimes  when  a  very  large  sur- 
face, such  as  the  peritoneum,  is  suddenly  involved. 

Centanni1  obtained  through  warmth  and  alcohol 
from  the  bodies  of  bacteria  a  substance  called  pyro- 
toxin,  which  was  with  difficulty  dialyzed.  From  dif- 
ferent bacteria  not  only  the  physiological  but  also  the 
chemical  properties  of  the  pyrotoxin  were  the  same. 
Not  only  did  this  cause  fever,  but  also,  when  persisted 
in,  it  produced  emaciation,  quickened  heart-action, 
apathy,  dyspnoea,  etc.  0 

The  bacterial  poisons  produce  an  increase  in  the 
number  of  leucocytes  and  a  lessening  in  the  amount  of 
haemoglobin  in  the  blood.  The  deleterious  effects  on 
the  nutrition  are  partly  due  to  the  direct  effect  of  the 
poison  and  partly  to  the  diseased  conditions  of  the 
organs  of  the  body,  such  as  the  spleen,  kidney,  and 
liver.  Degeneration  of  the  nerve  cells  is  frequently 
noticed  after  infectious  diseases;  especially  is  this  true 
of  diphtheria.  Several  bacterial  poisons  have  been 
found  to  produce  convulsions;  the  best  example  of  this 
is  the  tetanus  toxin. 

The  true  bacterial  poisons  are,  as  already  stated, 
neither  alkaloids  nor  albumins.  Some  of  them,  such 
as  the  diphtheria  and  tetanus  toxins,  are  peculiar  in 
their  effects,  while  others,  such  as  those  produced  by 
the  pneumococcus  and  streptococcus,  can  scarcely  be 
distinguished.  They  are  destroyed  by  heat  at  70°  C. 

1  Deutsche  med.  Wochenschrift,  1894,  Nos.  7  and  8. 


RELATION  OF  BACTERIA  TO  DISEASE.         95 

Bacteria  also  produce  secondary  poisons,  which  stand 
a  temperature  of  100°  to  120°  C. 

The  Influence  of  Quantity  in  Infection.  With  bacteria 
the  number  introduced  has  an  immense  influence  upon 
the  probability  of  infection  taking  place. 

If  we  introduce  into  a  culture  medium,  which,  like 
the  body,  is  only  fairly  suitable  for  growth,  a  few 
bacteria,  it  is  not  improbable  that  they  may  all  die; 
whereas  if  a  greater  number  are  introduced,  while  there 
will  at  first  be  a  slight  diminution  of  these,  those  that 
die  seem  to  neutralize  the  substances  which  were  dele- 
terious; then  those  bacteria  which  survive  begin  to  in- 
crease, and  soon  they  multiply  enormously.  The  same 
is  true  for  parasitic  bacteria  in  the  body.  A  few  only 
gaining  entrance,  they  may  die;  a  larger  number  being 
introduced,  some  may  or  may  not  survive  ;  but  if 
a  still  greater  quantity  is  injected  it  is  almost  certain 
that  there  will  be  some  surviving  members,  which,  after 
the  destruction  of  antagonistic  substances,  and  on  be- 
coming accustomed  to  their  environment,  will  begin  to 
grow  and  produce  disease. 

With  those  bacteria  whose  virulence  is  great — i.  e., 
those  which  are  capable  of  growing  with  great  ease  in 
the  body  fluids — a  very  few  organisms  will  produce 
disease  almost  as  quickly  as  a  million,  allowance  only 
being  made  for  the  short  time  required  for  the  few 
to  become  equal  in  number  to  the  million.  At  the 
other  extreme  of  virulence,  however,  many  millions 
may  have  to  be  introduced  to  permit  of  the  develop- 
ment of  any  of  the  organisms  in  the  body.  With  these 
bacteria  we  are  thus  able  to  produce  either  no  effect 
whatever,  a  local  effect,  or  in  some  cases  a  general  sep- 
ticaemia, by  regulating  the  amount  of  infection  intro- 


96  BACTERIOLOGY. 

duced.  In  the  majority  of  cases  in  man  the  number  of 
bacteria  received  is  comparatively  small;  but  by  the 
rupture  of  an  abscess  into  a  cavity  or  into  the  circula- 
tion, or  by  the  opening  of  the  intestinal  contents  into  the 
peritoneum,  the  quantity  introduced  may  be  enormous. 

The  Degree  of  Virulence  Possessed  by  Bacteria.  Bac- 
teria as  found  in  nature  differ,  as  has  already  been  stated, 
as  to  the  amount  of  poison  they  produce  and  the  ease  and 
rapidity  with  which  they  grow  in  any  nutritive  sub- 
stance. Both  of  these  properties  not  only  vary  greatly  in 
different  members  of  the  same  species,  but  each  variety 
of  bacteria  may  to  a  large  extent  be  increased  or  dimin- 
ished in  virulence.  The  specific  poisons  produced  by 
bacteria  can  be  best  studied  in  diphtheria  and  tetanus. 
We  note,  first,  that  different  individual  bacilli  of  diph- 
theria and  tetanus  have,  when  freshly  obtained,  wide 
variations  in  the  amount  of  toxin  which  they  produce 
— i.e.,  a  diphtheria  bacillus  obtained  from  a  case  of  diph- 
theria will  produce  in  suitable  nutrient  broth  a  poison 
of  such  strength  .that  1  c.c.  will  kill  an  average  sized 
guinea-pig,  while  the  poison  from  another  bacillus  will 
kill  with  a  much  less  quantity,  or  0.005  c.c.  Further, 
the  bacilli  obtained  from  some  sources  retain  their  power 
of  producing  poison,  when  grown  on  artificial  media,  for 
years  unaltered,  while  others  lose  much  of  this  in  a  few 
months.  This  is  equally  true  of  the  tetanus  bacilli. 

The  power  to  produce  toxin  can  be  taken  from  bacilli 
by  growing  them  under  adverse  circumstances,  such  as 
cultivation  at  the  maximum  temperature  at  which  they 
are  capable  of  development.  Some  bacilli  are  easily 
attenuated;  others  are  robbed  of  their  virulence  only 
with  great  difficulty.  Increase  of  toxin-production  is 
more  difficult,  and  it  is  only  possible  to  obtain  it  to  a 


RELATION  OF  BACTERIA  TO  DISEASE.        97 

certain  extent.  The  means  usually  employed  are  the 
frequent  replanting  of  cultures  and  their  growth  in  cap- 
sules placed  in  the  bodies  of  susceptible  animals.  But 
with  all  our  efforts  we  are  usually  only  able  to  restore 
approximately  the  degree  of  toxin-formation  which  the 
cultures  originally  possessed.  The  adaptation  of  bac- 
teria to  any  nutritive  substance,  living  or  dead,  so  that 
they  will  grow  more  readily,  is  more  easily  brought 
about,  provided  they  will  grow  at  all.  The  streptococcus 
from  erysipelas  and  the  pneumococcus  from  pneumonia 
are  typical  of  this  class  of  bacteria.  Inoculate  a  rabbit 
with  a  few  streptococci  obtained  from  a  case  of  human 
sepsis,  and,  as  a  rule,  no  result  follows  ;  inject  a  few 
million,  and  usually  a  local  induration  or  abscess  ap- 
pears ;  but  if  one  hundred  million  are  administered 
septicaemia  develops.  From  this  rabbit  now  inoculate 
another,  and  we  find  that  a  dose  slightly  smaller  suffices 
to  produce  the  same  effect;  in  the  next  animal  inoculated 
from  this  still  less  is  required,  and  so  on,  until  in  time, 
with  suitable  cultures,  a  very  minute  number  will  surely 
develop  and  produce  death.  The  same  increase  in 
virulence  can  be  noted  when  septic  infection  is  carried 
in  surgery  or  obstetrics  from  one  human  case  to  another. 
By  allowing  bacteria  to  continue  to  develop  under  cer- 
tain fixed  conditions  they  become  accustomed  to  them, 
and  less  adapted  for  all  that  differ. 

Somewhat  distinct,  again,  from  that  class  of  bacteria 
which  multiply  rapidly  are  those  which,  like  the  tubercle 
and  leprosy  bacilli,  develop  slowly.  Here  increase  of 
virulence  is  shown,  as  before,  by  the  production  of  dis- 
ease through  the  introduction  of  very  small  numbers 
into  the  body,  but  increase  in  rapidity  of  development 
cannot  progress  except  to  within  certain  limits.  A  sin- 

'  7 


98  BACTERIOLOGY. 

gle  streptococcus  may,  through  its  rapid  multiplication, 
produce  death  in  eighteen  hours  ;  a  single  tubercle 
bacillus,  on  the  other  hand,  cannot  produce  sufficient 
numbers  in  less  than  two  weeks.  The  virulence  of  the 
septicsemic  class  of  bacteria  is  not  at  all  the  same  when 
measured  in  different  animals,  and  it  is  largely  for  this 
reason  that  the  virulence  in  test  animals  does  not 
usually  correspond  with  the  severity  of  the  case  from 
which  the  organism  was  derived.  We  should  re- 
member in  this  connection  the  varying  power  of  resist- 
ance in  different  animals  and  of  the  same  individual 
at  different  times. 

Mixed  Infection.  The  combined  effect  upon  the  tissues 
of  the  products  of  two  or  more  varieties  of  pathogenic 
bacteria,  and  also  of  the  influence  of  these  different  forms 
on  each  other,  are  of  great  importance  in  the  produc- 
tion of  disease.  The  infection  from  several  different 
organisms  may  occur  at  the  same  time,  or  one  may  fol- 
low the  other  or  others — so-called  secondary  infection. 
Mixed  infection  arises  usually  from  the  inoculation  of 
more  than  one  variety  of  bacteria  simultaneously. 
Thus,  an  abscess  is  often  due  to  several  forms  of 
pyogenic  cocci.  If  a  wound  is  infected  from  such  a 
source  the  inflammation  produced  will  probably  be 
caused  by  all  the  varieties  present  in  the  original  in- 
fection. Peritonitis  following  intestinal  injuries  must 
necessarily  be  due  to  more  than  one  organism.  Thus, 
whenever  two  or  more  varieties  of  bacteria  are  trans- 
ferred to  a  new  soil,  mixed  infection  takes  place  if 
more  than  one  variety  is  capable  of  developing  in  that 
locality. 

Forms  of  infection  which  are  allied  to  both  mixed 
and  secondary  infection  are  those  occurring  in  the 


RELATION  OF  BACTERIA  TO  DISEASE.        99 

mucous  membranes  of  the  respiratory  and  digestive 
tract.  In  these  situations  pathogenic  bacteria  of  slight 
virulence  are  always  present  even  in  health.  Thus 
in  the  upper  air-passages  there  are  usually  found  strep- 
tococci, staphylococci,  andpneumococci.  When  through 
a  cold,  or  the  invasion  of  another  infective  agent,  as 
the  diphtheria  bacillus,  the  epithelium  of  the  mucous 
membrane  of  the  throat  is  injured  or  destroyed,  the  pyo- 
genic  cocci  already  present  are  now  enabled  in  this  dis- 
eased membrane  to  grow,  produce  their  poison,  and 
even  invade  deeper  tissues.  The  intestinal  mucous 
membrane  is  invaded  in  a  similar  way  by  the  colon 
bacilli  and  other  organisms  after  injury  by  the  typhoid 
bacilli  or  cholera  spirilla.  Generally  speaking,  all  in- 
flammations of  the  mucous  membranes  contain  some  of 
the  elements  of  mixed  infection.  Blood  infection,  on 
the  other  hand,  is  usually  due  to  one  form  of  bacteria, 
as  even  when  several  varieties  are  introduced,  only 
one,  as  a  rule,  is  capable  of  development.  The  same 
is  true  to  a  somewhat  less  extent  of  inflammation  of 
the  connective  tissue.  The  additional  poison  given  off 
by  the  associated  bacteria  aid  infection  by  causing  a 
lowering  of  the  vital  resistance  of  the  body. 

The  bacteria  are  also  at  times  directly  influenced  by 
the  products  of  associated  organisms.  These  may 
affect  them  injuriously,  as,  for  example,  the  pyogenic 
cocci  in  anthrax;  or  they  may  be  necessary  to  their 
development,  as  in  the  case  of  anaerobic  bacteria.  JSTot 
infrequently  the  tetanus  bacilli  or  spores  would  not  be 
able  to  develop  in  wounds  were  it  not  for  the  presence 
of  aerobic  bacteria  introduced  with  them.  This  is  shown 
outside  the  body,  where  tetanus  bacilli  will  not  grow  in 
the  presence  of  oxygen  unless  aerobic  bacteria  are  asso- 


100  BACTERIOLOGY. 

elated  with  them.  Again,  it  is  found  that  the  associa- 
tion of  one  variety  with  another  may  increase  its  viru- 
lence. Thus  Roux  and  Yersiu  believe  that  they  have  es- 
tablished the  fact  that  streptococci  and  diphtheria  bacilli 
mutually  increase  each  other's  virulence.  On  the  other 
hand,  the  absorption  of  the  products  of  certain  bacteria 
immunizes  the  body  against  the  invasion  of  other  bac- 
teria, as  shown  by  Pasteur  that  attenuated  chicken 
cholera  cultures  produce  immunity  against  anthrax. 

The  Modes  of  Entrance  of  Infection.  The  various  fluids 
and  tissues  of  the  body  differ  greatly  in  their  chemical 
constituents,  their  reaction,  their  protection  from  in- 
fection, their  access  to  free  oxygen,  their  temperature, 
and  in  other  less  well-known  respects.  These  varia- 
tions are  sufficient  to  render  certain  portions  of  the 
body  suitable  for  the  growth  of  some  bacteria  and 
unsuitable  for  others.  This  fact  is  of  immense  im- 
portance in  the  transmission  or  prevention  of  disease. 
Thus,  for  example,  let  us  rub  very  virulent  strepto- 
cocci, typhoid  bacilli,  and  diphtheria  bacilli  into  an 
abrasion  on  the  hand.  The  typhoid  bacillus  produces 
no  lesion,  the  diphtheria  bacillus  but  a  very  minute 
infected  area,  but  the  streptococcus  gives  rise  to  a  severe 
cellulitis  or  fatal  septicaemia.  Now  place  the  same 
bacteria  on  an  abrasion  in  the  throat.  The  typhoid 
bacillus  is  again  harmless;  the  diphtheria  bacillus  pro- 
duces inflammation,  a  pseudomembrane,  and  toxaemia, 
and  the  streptococcus  causes  an  exudate,  an  abscess,  or 
a  septicaemia.  Finally,  introduce  the  same  bacteria  into 
the  intestines,  and  now  it  is  the  typhoid  bacillus  which 
produces  its  characteristic  lesions,  while  the  strepto- 
coccus and  diphtheria  bacillus  are  usually  innocuous. 

If  we  tried  in  this  way  all  the  parasitic  bacteria  we 


RELATION  OF  BACTERIA  TO  DISEASE.       1Q1 

would  find  that  certain  varieties  are  capable  of  develop- 
ing and  thereby  producing  disease  only  on  the  mucous 
membrane  of  the  throat,  others  of  the  intestine,  others 
of  the  urethra  ;  some  develop  only  in  a  wound  or  in 
the  blood,  while  others,  again,  under  favorable  condi- 
tions, seem  able  to  grow  in  or  upon  almost  any  region 
of  the  body. 


CHAPTER  V. 

IMMUNITY. 

THAT  certain  races  of  animals  and  men,  and  certain 
individuals  among  these,  are  more  refractory  to  disease 
than  others,  is  a  fact  which  has  long  been  known. 
Experience  and  observation  have  taught  us,  further, 
that  the  same  individuals  are  at  one  time  more  resistant 
to  disease  than  at  another.  This  inborn  or  spontaneous 
refractory  condition  is  termed  natural  immunity,  in  con- 
tradistinction to  that  acquired  by  recovery  from  disease. 

As  in  bacteria,  we  distinguish  between  the  ability  to 
produce  poison  and  the  power  to  multiply  in  the  body, 
so  here  we  may  distinguish  between  immunity  to  poison 
and  immunity  to  the  development  of  bacteria. 

With  regard  to  variations  in  susceptibility,  certain 
known  facts  have  been  ascertained.  Thus,  cold-blooded 
animals  are  generally  insusceptible  to  infection  from 
those  bacteria  which  produce  disease  in  warm-blooded 
animals,  and  vice  versa.  This  is  readily  explained  by 
the  inability  of  the  bacteria  which  grow  at  the  tem- 
perature of  warm-blooded  animals  to  thrive  at  the 
temperature  existing  in  cold-blooded  animals.  But  dif- 
ferences are  observed  not  only  between  warm-blooded 
and  cold-blooded  animals,  but  also  between  the  several 
races  of  warm-blooded  animals.  The  anthrax  bacillus 
is  very  infectious  for  the  mouse  and  guinea-pig,  while 
the  rat  is  not  susceptible  to  it  unless  its  body  resistance 


IMMUNITY.  103 

is  reduced  by  disease  and  the  amount  of  infection  is 
great.  The  inability  of  a  micro-organism  to  grow 
in  the  body  of  an  animal  does  not  usually  indicate, 
however,  an  insusceptibility  to  its  poison;  thus,  for  in- 
stance, rabbits  are  less  susceptible  than  dogs  to  the  effects 
of  the  poison  elaborated  by  the  pneumococci,  but  these 
bacteria  develop  much  better  in  the  former  than  in 
the  latter.  Differences  in  susceptibility  are  sometimes 
very  marked  among  different  varieties  of  the  same  race 
of  animals,  as,  for  instance,  between  different  kinds  of 
rats  and  pigeons  to  anthrax.  In  animals,  as  a  whole, 
it  is  noticed  experimentally  that  the  young  of  all  species 
are  less  resistant  to  infection  than  the  older  and  larger 
ones. 

The  difficulty  experienced  by  the  large  majority  of 
bacteria  in  developing  in  the  tissues  of  the  healthy 
body  can  be  to  a  great  extent  removed  by  any  cause 
which  lowers  the  general  or  local  vitality  of  the  tissues. 
Among  the  causes  which  bring  about  such  lessened  re- 
sistance of  the  body  are  hunger  and  starvation,  bad 
hygienic  surroundings,  exhaustion  from  overexertion, 
exposure  to  cold,  the  deleterious  effects  of  poisons,  bac- 
terial or  other,  acute  and  chronic  diseases,  vicious 
habits,  drunkenness,  etc.  Purely  local  injuries,  such 
as  wounds,  contusions,  etc.,  also  give  sometimes  a  point 
of  entrance  for  infection,  or  at  least  a  point  of  less  re- 
sistance, where  the  bacteria  may  develop  and  produce 
local  inflammation.  This  is  noted  in  infection  by  the 
tubercle  and  typhoid  bacilli,  pyogenic  cocci,  etc. 
Local  affections,  such  as  endocarditis,  may  also  afford 
a  weak  spot  for  the  bacteria  to  seize  upon.  The  pres- 
ence of  foreign  bodies  in  the  tissues  in  like  manner 
predisposes  them  to  bacterial  invasion.  Interference 


104  BACTERIOLOGY. 

with  free  circulation  of  blood  and  retention  in  the  body 
of  substances  which  should  be  eliminated  also  tend  to 
lessen  the  vitality.  In  these  and  other  similar  ways 
animals  which  are  otherwise  refractory  may  acquire  a 
susceptibility  to  disease. 

Immunization  and  Healing  by  Non-specific  Means.  Just 
as  all  conditions  which  are  deleterious  to  the  body  lessen 
its  power  of  resistance  to  bacterial  invasion,  so  all  con- 
ditions which  are  favorable  to  it  increase  its  resistance, 
and  thus  aid  in  preventing  and  overcoming  infection. 
The  internal  use  of  antiseptics  against  bacteria  has 
not  proved  successful,  for  the  reason  that  an  amount 
too  small  to  inhibit  bacterial  growth  is  found  to  be 
poisonous  to  the  tissue  cells.  The  efficacy  of  quinine 
in  malaria  and  mercury  in  syphilis  is,  possibly,  an  ex- 
ception to  the  rule,  but  in  both  cases  we  are  dealing 
probably  with  animal  parasites,  not  ordinary  bacteria. 
Such  substances  as  nuclein  and  others  contained  in 
blood-serum,  when  introduced  into  the  body  in  consider- 
able quantity,  aid  somewhat  in  inhibiting  or  preventing 
the  growth  of  many  bacteria.  Even  bouillon,  salt 
solution,  and  small  amounts  of  urine  have  a  slight  in- 
hibitory action.  The  hastening  of  elimination  of  the 
bacterial  poisons  by  free  intestinal  evacuation  and  en- 
couragement of  the  functions  of  the  skin  and  kidneys 
are  also  of  some  avail.  The  enzymes  formed  by  certain 
bacteria  have  been  found  to  exert  a  slight  bactericidal 
action,  not  only  on  the  germs  which  have  directly  or 
indirectly  produced  them  in  the  body,  but  also  on  other 
varieties.  JSTone  of  these  enzymes  are  sufficiently  pro- 
tective to  be  of  practical  value  nor  equal  in  power  to  the 
protective  substances  formed  by  the  tissues  from  the 
bacterial  products. 


IMMUNITY.  105 

The  Use  of  Local  Treatment  in  Inhibiting  Bacterial  In- 
vasion. The  total  extirpation  of  the  infected  area  by 
surgical  means,  if  thoroughly  carried  out,  removes  the 
disease  entirely;  but,  unfortunately,  this  procedure  is 
rarely  possible.  When  incomplete  it  is  frequently 
helpful;  but  it  may  be  harmful,  for  by  creating  and 
exposing  fresh  wounded  surfaces  to  infection  it  may 
lead  to  the  further  development  of  the  disease.  Again, 
it  may  be  useless,  for  by  removing  only  a  portion  of 
the  bacteria  it  may  leave  those  which  have  already 
reached  the  deeper  tissues  or  blood  to  go  on  devel- 
oping. In  some  cases,  like  anthrax  and  infection 
from  bites  of  rabid  animals,  total  removal  of  the 
virus  is  possible,  either  by  the  knife  or  thorough  cauter- 
ization, and  will  prevent  a  general  infection.  So  also 
in  tetanus,  the  invasion  being  limited,  surgical  inter- 
ference may  be  of  great  use  by  removing  not  only  the 
bacilli  themselves  but  also  that  portion  of  their  poison 
which  has  not  as  yet  been  absorbed  from  the  tissues. 
The  beneficial  effects  of  opening  an  abscess,  incising  a 
cellulitis,  or  cleansing  and  drainage  of  the  uterine 
cavity  are  well  known.  The  retention  of  the  poisonous 
products  of  the  bacteria  and  altered  tissue  substance 
leads  to  their  absorption,  and  thus  lowers  the  tone  of 
the  neighboring,  and  to  a  less  extent  of  the  general, 
tissues  in  consequence  of  the  poisoning.  This  enables 
the  bacteria  to  penetrate  into  tissues  which  would  other- 
wise resist  them.  The  mechanical  effect  of  pressure  on 
the  walls  of  an  abscess  by  its  contents  also  aids  the  bac- 
terial progress.  Local  bleeding  and  the  application  of 
cold  probably  act  by  lessening  tension.  The  application 
of  warmth  hastens  absorption,  and  so,  when  the  infec- 
tion is  one  which  tends  to  localize,  it  acts  favorably  by 


106  BACTERIOLOGY. 

accelerating  the  development  and  thus  the  disappear- 
ance of  the  inflammation.  A  peculiar  effect  of  opera- 
tive interference  is  noticed  in  the  frequently  observed 
beneficial  result  of  laparotomy  in  tubercular  peritonitis. 

Antiseptic  solutions  have  the  power  of  cleansing  and 
rendering  sterile  the  surfaces  of  a  wound — that  is,  of 
preventing  the  introduction  of  infection.  After  infec- 
tion has  taken  place,  however,  it  is  doubtful  whether 
antiseptic  washing  has  much  more  direct  influence 
than  simple  cleansing,  and  it  certainly  can  have  no 
bactericidal  effect  at  any  distance  from  the  surface, 
either  direct  or  indirect.  Certain  infectious  diseases 
which  are  comparatively  superficial  are  probably  bene- 
fited by  antiseptic  solutions,  such  as  gonorrhoea,  diph- 
theria, and  other  inflammations  of  the  mucous  mem- 
branes. Even  here,  however,  it  is  impossible  to  do  more 
than  disinfect  superficially,  and  in  some  cases  any 
irritation  of  the  tissues  is  apt  to  do  more  harm  than 
good.  In  the  superficial  lesions  of  syphilis  and  tuber- 
culosis the  local  use  of  antiseptics  is  sometimes  of 
great  value.  In  these  diseases  the  irritant  effects  of  the 
antiseptics  which  stimulate  the  tissues  may  also  be 
beneficial.  Roux  has  reported  that  certain  specific 
serums,  just  as  certain  enzymes,  have  some  destructive 
effect  on  the  toxic  substances  of  other  species  of  bac- 
teria; but  this  is  a  subject  which  has  been  as  yet  but 
little  investigated. 

Specific  immunity r,  or  a  condition  of  the  body  which 
prevents  the  development  in  it  of  one  variety  of  micro- 
organisms and  renders  it  unaffected  by  their  bacterial 
poisons.  The  invasion  of  the  body  with  more  or  less 
serious  results  by  most  micro-organisms  is  followed 
by  a  condition  which  for  a  variable  period  and  to  a 


IMMUNITY.  107 

variable  degree  is  deleterious  to  their  further  growth. 
It  also  gives  rise  to  substances  which  neutralize  the 
poisonous  effects  of  the  bacterial  products.  This  im- 
munity may  take  place  in  various  ways  : 

1.  Through    recovery   from    disease   naturally  con- 
tracted or  from  infection  artificially  produced.     This 
immunity   may   be    slight,    as    after    recovery    from 
erysipelas  or  pneumonia,  marked  for  a  short  period  of 
time,  as  in  diphtheria  and  typhoid  fever,  or  prolonged, 
as  after  scarlet  fever  or  syphilis. 

2.  By  the  injection  of  the  bacteria  into  tissues  not 
well  suited  to  their  development,  as  the  injection  of 
typhoid  bacilli  or  cholera  spirilla  into  the  subcutaneous 
tissues.     Here  a  mild  local  infection  follows,  with  con- 
siderable resulting  immunity. 

3.  By  the  injection  of   micro-organisms  attenuated 
by  heat,  chemicals,  or  other   means.     In  this  case  a 
local  or  general  infection  of  the  animal  is  produced, 
of  moderate  severity,  as  a  rule,  and  the  immunity  is  not 
as  marked  and  lasting  as  after  recovery  from  a  more 
serious   attack  ;    but   it   is,  nevertheless,  considerable. 
The  inoculation  of  sheep  with  the  attenuated  anthrax 
bacillus  and  the  use  of  vaccination  in  man  are  examples 
of  this  method. 

4.  By  the  injection  of  the  unaltered  chemical  con- 
stituents of    the  dead   bodies  of  bacteria  and  of   the 
chemical  products  which  they  elaborate  and  discharge 
into  the  surrounding  culture  media  during  life.    Smith 
and  Salmon  proved  that  by  repeated  injections  of  the 
filtered  bouillon  cultures  of  the  hog-cholera  bacillus  a 
considerable  immunity  may  be  produced  against  the  in- 
vasion of  this  bacillus.     Similar  results  have  followed 
the  injections  of  dead  cultures  of  typhoid  and  anthrax 


108  BACTERIOLOGY. 

bacilli  and  cholera  spirilla,  etc.  After  infection  with 
most  parasitic  bacteria  the  body  resistance  to  the  growth 
of  the  same  organism  is  greatly  increased;  in  other  in- 
fections, however,  it  is  but  slightly  augmented. 

The  protective  substances  held  in  solution  in  the 
blood-serum  are  clearly  apparent  in  their  effects  either 
in  preventing  the  increase  of  the  bacteria  or  neutralizing 
the  toxic  action  of  their  products;  chemically,  however, 
they  are  but  little  understood,  and  although  some  of 
them  have  been  shown  to  be  to  a  large  extent  specific, 
that  is,  they  are  far  more  efficient  in  protecting  against 
the  special  variety  of  bacteria  which  produced  the  infec- 
tion than  against  any  other,  still  we  have  no  knowledge 
of  any  chemical  difference  between  them.  The  addition 
of  0.5  per  cent,  of  carbolic  acid  injures  these  substances 
but  slightly.  At  ordinary  temperatures  there  is  a 
gradual  deterioration  in  value,  so  that  in  from  one  to 
six  months  they  may  become  inert.  Twenty  hours' 
exposure  to  a  temperature  of  60°  C.  does  not  destroy 
them,  but  one  hour  at  70°  C.  does  so  almost  totally. 
Different  protective  substances  differ  as  to  the  rapidity 
with  which  they  deteriorate. 

Suitable  animals  after  repeated  infections  gradually 
accumulate  in  their  blood  considerable  amounts  of  these 
protective  substances,  so  that  very  small  amounts  of 
serum  will  inhibit  the  growth  of  the  bacteria  or  neu- 
tralize their  products.  Thus,  0.1  c.c.  of  a  serum  from 
a  horse  frequently  infected  by  the  pneumococcus  will 
prevent  the  development  in  the  body  of  a  rabbit  of  one 
hundred  times  the  fatal  dose  of  very  virulent  pneumo- 
cocci,  and  a  few  times  a  fatal  dose  of  less  virulent  ones, 
the  actual  number  as  well  as  the  virulence  of  the  bac- 
teria affecting  the  protective  value  of  the  serum. 


IMMUNITY.  109 

These  protective  substances  are  found  also  in  other 
fluids  of  the  body  than  in  the  blood;  they  occur,  in- 
deed, in  the  substance  of  all  cells  to  a  greater  or  less 
extent.  How  much  of  this  is  simply  in  solution  from 
the  serum,  and  where  the  substances  are  formed,  is  not 
definitely  known. 

5.  By  the  injection  of  the  blood-serum  of  animals 
which  have  previously  passed  through  a  specific  disease 
or  have  been  inoculated  with  the  bacterial  products. 
The  first,  probably,  to  think  of  the  possibility  of  effect- 
ing this  was  Raynaud,  who,  in  1877,  showed  that  the 
injection  of  large  quantities  of  serum  derived  from  a 
vaccinated  calf  into  an  animal  prevented  its  successful 
vaccination.  Hericourt,  Richet,  and  others  demon- 
strated the  same  thing  for  other  diseases.  The  results 
obtained  by  Behring  and  Kitasato  upon  diphtheria  and 
tetanus,  where,  indeed,  the  serum  prevented  the  action 
of  the  poisons  rather  than  the  direct  development  of  the 
bacteria,  gave  a  still  greater  impetus  to  these  investiga- 
tions. 

The  immunity  produced  by  these  substances  affects 
the  entire  body,  as  is  only  natural,  since  the  blood  into 
which  they  are  absorbed  is  distributed  everywhere. 
When  the  immunity  is  but  slight,  infection  may  take 
place  in  the  more  sensitive  regions  and  still  be  im- 
possible in  those  tissues  having  more  natural  resistance. 
If  the  serum  is  injected  into  other  animals  or  man  the 
immunity  is  greatest  immediately  after  absorption,  and 
then  declines,  being  rather  quickly  (in  several  weeks  or 
months),  almost  entirely  lost,  so  that  repeated  injections 
are  required  to  maintain  the  immunity.  This  is  dis- 
tinctly in  contrast  to  the  immunity  acquired  after  the 
introduction  of  bacterial  products,  where  the  tissues 


HO  BACTERIOLOGY. 

of  the  organism,  in  ways  unknown,  give  out,  in  response 
to  the  bacterial  stimulus,  inhibitory  or  antitoxic  sub- 
stances, or  combine  with  the  bacterial  poisons  to  produce 
them.  Here  immunity  reaches  its  height  a  week  or  ten 
days  after  the  injection,  and  then  continues  for  a  week 
or  two,  when  it  slowly  declines  again.  The  serum  im- 
munity is  frequently  called  passive  immunity  and  the 
bacterial  immunity  active  immunity. 

If  a  greater  quantity  of  protective  substance  is  de- 
sired in  the  blood  than  occurs  after  one  infection,  re- 
peated injections  of  living  or  dead  bacteria  and  their 
products  are  given,  the  doses  being  administered  at 
short  intervals  and  in  sufficient  amount  to  produce  a 
slight  elevation  of  temperature  and  malaise.  Then,  as 
soon  as  the  animal  returns  to  a  normal  condition,  another 
injection  of  slightly  greater  quantity  is  given.  After 
several  months  of  such  treatment  the  blood  is  withdrawn, 
allowed  to  clot,  and  the  serum  then  siphoned  off  asep- 
tically  and  stored  either  with  or  without  the  addition  of 
preservatives.  The  serum  is  tested  by  mixing  it  with  a 
certain  number  of  times  the  fatal  dose  of  a  culture  or  its 
toxins  whose  virulence  or  toxicity  is  known,  and  then 
injecting  this  under  the  skin,  in  the  vein,  or  into  the 
peritoneum,  according  to  the  nature  of  the  bacteria  to 
be  tested.  The  main  point  is  that  some  definite  method 
be  carried  out  by  which  the  relative  value  of  the  serum 
can  be  judged  in  comparison  with  other  serums.  As  a 
rule,  the  value  is  stated  in  the  number  of  fatal  doses  of 
culture  or  toxin  which  a  fraction  of  a  cubic  centimetre 
of  serum  will  prevent  from  destroying  the  animal.  It 
is  well  to  remember,  that  with  a  living  germ  a  mul- 
tiple of  a  fatal  dose  is  not  as  much  more  severe  than  a 
single  dose  as  the  figure  would  suggest.  One  thousand 


IMMUNITY.  HI 

times  a  fatal  dose  of  a  very  virulent  micro-organism 
will  be  neutralized  by  several  times  the  amount  of 
serum  which  a  single  fatal  dose  requires,  since  in  the 
case  of  very  virulent  living  bacteria  whose  virulence 
is  due  to  their  ability  to  increase,  it  is  not  the  organ- 
isms which  are  introduced  that  kill  but  the  millions 
that  develop  from  them.  As  a  rule,  the  serum  has  to 
be  given  before  the  bacteria  introduced  into  the  body 
have  multiplied  greatly.  After  that  period  has  elapsed 
the  serum  usually  fails  to  act,  but  some  serums  will 
prevent  further  development  even  then.  The  immu- 
nity conferred  on  a  person  from  serum  lasts  from  a 
few  days  to  several  months,  according  to  the  amount 
of  serum  injected.  As  in  animals,  it  is  strongest 
immediately  after  absorption.  An  injection  of  bac- 
terial poisons  or  the  contraction  of  actual  disease  usu- 
ally confers  immunity  from  one  to  three  weeks  after 
the  infection,  and  lasts,  according  to  the  nature  of  the 
infection,  from  one  month  to  a  year  or  more.  The 
serum  loses  all  appreciable  protective  value  as  measured 
in  test  animals  in  the  usual  doses  before  the  person  is 
liable  to  infection.  Repeated  injections  of  serum  con- 
tinue this  condition  of  immunity  indefinitely. 

The  use  of  serums  having  specific  protective  proper- 
ties has  been  tried  both  in  animals  and  man  as  a  pre- 
ventive of  infection.  In  susceptible  animals  injections 
of  some  of  the  very  virulent  bacteria,  as  pneumococci, 
streptococci,  typhoid  bacilli,  and  cholera  spirilla,  can 
be  robbed  of  all  danger  if  small  doses  of  their  re- 
spective serums  are  given  before  the  bacteria  have 
increased  to  any  great  extent  in  the  body.  If  given 
later  they  are  ineffective.  For  some  bacteria,  such 
as  tubercle  bacilli,  no  serum  has  been  obtained  of  suffi- 


112  BACTERIOLOGY. 

cleat  power  to  prevent  infection.  Through  serums, 
therefore,  we  can  immunize  against  an  infection,  and 
even  stop  one  just  commencing;  but  as  yet  we  cannot 
cure  an  infection  which  is  already  fully  developed, 
though  even  here  there  is  reason  to  believe  that  we 
may  possibly  prevent  an  invasion  of  the  general  system 
from  a  diseased  organ  as  by  the  pneumococcus  from 
an  infected  lung  in  pneumonia.  On  the  whole,  the 
serums  which  simply  inhibit  the  growth  of  bacteria 
have  not  given,  as  observed  in  practice,  conclusive 
evidence  of  great  value  in  already  developed  disease. 
This  is  partly  due  to  the  difficulty  to  be  discussed  fully 
later  of  determining  early  enough  the  exact  nature  of 
the  bacteria  causing  the  infection. 

Acquired  Immunity  to  Poison.  Although  the  serum 
of  animals  which  have  been  infected  with  any  one  of 
many  varieties  of  bacteria  is  usually  both  antitoxic  and 
bactericidal,  still  one  of  these  protective  substances  may 
be  present  almost  alone;  thus  antitoxic  substances  are 
present  almost  exclusively  in  animals  injected  with  two 
species  of  bacteria  which  produce  powerful  specific 
poisons — viz.,  the  bacilli  of  diphtheria  and  tetanus. 
When  the  toxins  of  either  of  these  are  injected  in 
small  amounts  the  animals  after  complete  recovery 
are  able  to  bear  a  larger  dose  without  deleterious 
effects,  and  these  doses  in  the  more  suitable  animals 
can  be  gradually  increased  until  a  thousand  times  a 
previously  fatal  dose  may  be  administered  without  any 
serious  results  whatever.  To  Behring  and  Kitasato  we 
owe  the  discovery  that  this  protecting  substance  accu- 
mulates to  such  an  extent  in  the  blood  that  very  small 
amounts  of  serum  are  sufficient  to  protect  other  animals 
from  the  effects  of  the  toxin. 


IMMUNITY.  113 

Some  other  important  parasitic  bacteria  produce 
toxins  and  in  the  body  antitoxins,  but  all  to  a  far  less 
extent  than  those  of  tetanus  and  diphtheria.  Follow- 
ing them  is  the  plague  bacillus,  and  then,  but  far 
behind,  the  cholera  spirilla,  the  typhoid  bacilli,  the 
streptococci,  etc.  These  latter  bacteria  produce  more 
of  the  substances  which  inhibit  bacterial  growth  than 
of  those  which  neutralize  their  toxins. 

The  effect  of  the  antitoxin  is  to  prevent  the  poison- 
ous action  of  the  toxin.  It  does  not,  so  far  as  we 
know,  influence  the  cells  after  they  have  been  injured 
by  the  toxin;  it  is,  therefore,  a  preventive  rather  than 
a  cure.  \Ve  find,  experimentally,  that  a  very  much 
smaller  amount  of  antitoxin  will  neutralize  a  fatal  dose 
of  toxin  in  an  animal,  if  given  before  or  at  the  same 
time,  than  if  given  only  shortly  after  it.  An  animal 
already  profoundly  poisoned  by  the  toxin  is  unaffected 
by  any  amount  of  antitoxin. 

The  antitoxins  of  diphtheria  and  tetanus  are  gradu- 
ally eliminated  from  the  body  after  their  injection  or 
after  their  production  from  toxin  injections.  After 
the  usual  immunizing  dose  the  duration  of  immunity 
is  only  from  two  to  six  weeks,  the  period  differing  in 
each  individual.  The  elimination  of  the  antitoxin 
takes  place  partly  through  the  urine  and  other  secre- 
tions, and  it  is  partly  destroyed  in  the  body.  An 
animal  which  has  been  highly  immunized  will  retain 
considerable  amounts  of  antitoxin  for  from  two  to  four 
months. 

The  antitoxins  as  contained  in  the  serum  are  fairly 
stable.  The  different  antitoxins  vary  thus,  that  of 
diphtheria  is  somewhat  more  stable  than  that  of  tetanus. 
Kept  aseptically  in  cold  and  dark  storage,  and  pro- 

8 


114  BACTERIOLOGY. 

tected  from  access  of  air,  the  more  resistant  antitoxins 
may  be  preserved  sometimes  for  a  year  or  two  with 
practically  no  deterioration  in  strength.  At  other 
times,  however,  from  unknown  causes,  they  are  gradu- 
ally destroyed,  so  that  there  may  be  a  loss  of  about  10 
per  cent,  per  month.  A  serum  requires,  therefore,  to  be 
tested  every  few  months  if  we  wish  to  be  assured  of  its 
strength  in  antitoxin.  Preservatives,  such  as  carbolic 
acid,  trikresol,  camphor,  etc.,  alter  antitoxins  only  very 
slightly  when  in  dilute  solution,  but  in  strong  solution 
they  partially  destroy  them.  Heat  up  to  62°  C.  does 
not  injure  them  greatly,  but  higher  temperatures  alter 
them.  In  animals  injected  with  diphtheria  toxin 
Atkinson  has  found  that  there  is  with  the  increase  in 
antitoxin  an  almost  proportional  increase  in  globulin.1 
He  also  found  that  the  antitoxins  behave  like  globulins 
with  the  various  reagents,  being  completely  precipi- 
tated by  magnesium  sulphate.  NaCl,  when  added  to 
saturation  to  globulin  solutions  holding  antitoxin,  parti- 
ally precipitates  the  globulin  and  the  antitoxin.  When 
raised  to  72°  C.  a  series  of  precipitations  are  obtained 
which  contain  at  least  the  greater  part  of  the  antitoxin. 
Whether  this  indicates  that  antitoxin  is  a  form  of 
globulin  or  merely  that  it  is  similarly  affected  by  many 
reagents,  and  that  the  toxin  in  some  way  stimulates  the 
development  of  both,  it  is  as  yet  impossible  to  say. 
Although  in  a  very  rough  way,  the  same  animal  pro- 
duces antitoxin  in  direct  proportion  to  the  amount  of 
toxin  injected  so  long  as  its  condition  remains  good,  yet 
different  animals  of  the  same  species  give  very  varying 
amounts  from  the  same  injections,  some  not  giving  one- 

1  This  work,  carried  out  in  the  Research  Laboratory  of  the  Department  of 
Health  of  New  York  City,  will  appear  in  the  Journ.  of  Exp.  Med.  in  1900. 


IMMUNITY.  115 

fourth  of  that  furnished  by  others.  The  antitoxin  pro- 
duced by  a  certain  number  of  fatal  doses  of  toxin  will 
neutralize  many  thousand  times  that  amount. 

Diphtheria  and  tetanus  antitoxin  are  measured  by  the 
protective  power  of  the  serum  in  which  they  are  in 
solution — that  is,  the  amount  of  serum  required  to  pro- 
tect susceptible  animals  from  a  certain  number  of  fatal 
doses.  In  diphtheria  this  is  measured  in  units  ;  in 
tetanus  usually  by  the  proportion  which  exists  between 
the  amount  of  serum  used  and  the  animal's  weight.  For 
detailed  information,  see  under  Diphtheria  and  Tetanus. 

Antitoxins  are  absorbed  to  a  very  slight  extent  only 
when  taken  by  the  mouth — certainly  less  than  5  per 
cent.  They  must,  therefore,  be  introduced  subcuta- 
neously  or  intravenously  to  enter  the  body.  The  anti- 
toxic serum  does  not  act  against  the  bacteria  directly, 
but  by  neutralizing  their  poison,  it  prevents  them  from 
acting  as  irritants  to  the  cells,  and  so  the  soil  for  the 
growth  of  the  bacteria  becomes  unsuitable,  and  they 
cease  to  develop.  The  diphtheria  bacilli  grow  perfectly 
well  iu  their  antitoxic  serum. 

The  Elimination  of  the  Bacteria  and  Their  Products. 
This  takes  place  by  the  direct  separation  and  removal 
of  the  bacteria  where  there  is  access  to  the  outside, 
such  as  exists  in  the  mucous  membranes  of  the  respi- 
ratory, digestive,  and  urinary  tracts,  and  from  the 
cutaneous  surfaces,  etc.  The  elimination  of  the  bac- 
teria and  their  products  is  almost  a  necessity  where 
there  has  been  any  great  accumulation,  if  healing 
occurs.  When  the  bacteria  have  penetrated  deeply 
into  the  tissues,  and  continue  steadily  advancing,  the 
elimination  from  the  surface  is  of  little  curative  value, 
as  the  number  thrown  off  is  so  small  in  comparison 


116  BACTERIOLOGY. 

with  the  number  remaining.  Occasionally  the  casting 
off  of  the  bacteria  allows  them  to  iufect  other  places, 
as  in  some  cases  where  laryngeal  and  intestinal  tubercu- 
losis follows  pulmonary  tuberculosis.  We  must  bear  in 
mind,  however,  that  infection  in  these  regions  may  have 
been  produced  through  the  lymph  and  blood  channels. 

In  nearly  all  cases  of  infection  the  products  of  bacte- 
rial growth  are  absorbed  into  the  blood,  and  along  with 
them  a  few  bacteria  also,  even  when  they  do  not  repro- 
duce themselves  in  it.  The  greater  the  extent  of  the 
infection  and  the  more  deep-seated  it  is  the  greater  is 
the  amount  of  absorption.  The  bacteria  enter  the  blood, 
according  toKruse,  by  (1)  passive  eutrance  through  the 
stomata  of  the  capillary  walls;  (2)  carriage  into  the 
blood  in  the  bodies  of  leucocytes;  (3)  growth  of  the 
bacteria  through  the  walls  of  the  vessels;  (4)  transmis- 
sion of  the  bacteria  through  the  lymph-glands  placed 
between  the  lymph  and  bloodvessels. 

When  bacteria  are  abundant  in  the  blood  they  become 
fixed  in  the  capillaries  of  one  or  all  of  the  organs,  espe- 
cially of  the  liver,  kidneys,  spleen,  and  lungs,  and  then, 
by  means  of  the  leucocytes,  which  penetrate  the  capil- 
lary walls,  or,  directly,  they  pass  into  the  tissues  and 
substance  of  the  organs.  They  thus  reach  the  lymph 
channels  and  glands,  or  through  the  secretions  gain 
entrance  into  the  gall-bladder,  saliva,  etc.,  or  press 
through  the  epithelium,  as  in  the  alveoli  of  the  lungs; 
more  rarely  they  pass  through  the  excretions  into  the 
urine,  as  in  typhoid  fever,  though  some  deny  that  this 
can  happen  unless  there  is  a  previous  inflammation  of 
the  kidneys.  The  passage  of  bacteria  through  the 
breast  is  important,  from  the  fact  that  milk  is  so 
largely  used  as  food.  Many  observers  have  reported 


IMMUNITY.  117 

the  finding  of  tubercle  bacilli  in  milk  when  the  gland 
itself  was  intact  and  the  animal  tubercular.  Some 
authorities  have  put  its  presence  in  milk,  under  these 
circumstances,  as  high  as  50  per  cent,  of  the  cases. 
This,  in  our  experience,  is  undoubtedly  too  high,  and 
probably  these  observers  have  been  deceived  by  the 
pseudo-tubercle  bacilli.  They  are  undoubtedly  pres- 
ent, however,  in  the  milk  of  some  animals  in  which 
tubercular  disease  of  the  gland  could  not  be  demon- 
strated. The  finding  of  streptococci  and  staphylococci 
is  due  probably  in  the  majority  of  cases  to  the  infections 
taking  place  as  the  milk  is  voided,  for  the  epithelium 
at  the  outlet  of  the  lacteal  ducts  is  always  infected  with 
staphylococci,  and  usually  streptococci,  which  have  often 
been  received  from  the  mouth  of  the  sucking  infant. 

Whether  bacteria  are  eliminated  from  the  blood  by 
the  sweat  is  a  mooted  point.  The  skin  is  always  the 
seat  of  the  staphylococcus  and  frequently  of  other  bac- 
teria, so  that  it  is  difficult  to  determine  in  any  given 
case  the  origin  of  the  bacteria  found  in  the  sweat. 
Many  observers  have  reported  the  passage  of  bacteria 
from  the  blood  through  the  mucous  membrane.  So 
long  as  the  organs  of  secretion  are  not  injured  it  is  not 
likely  that  many  micro-organisms  are  eliminated  from 
the  blood  in  this  way.  Bacteria  are  sometimes  elimi- 
nated through  the  urine,  but  here,  as  a  rule,  when 
great  numbers  of  organisms  are  found,  it  is  due  to 
development  in  the  bladder.  Such  removal,  moreover, 
has  little  if  any  beneficial  effect;  but,  on  the  other  hand, 
may  be  a  source  of  danger  to  others,  as  in  typhoid 
fever.  The  removal  of  the  poisonous  products  of  bac- 
teria by  the  kidneys,  intestines,  etc.,  on  the  contrary, 
is  of  great  advantage  to  the  organism. 


CHAPTER   VI. 

THEORIES  OF  INFECTION,   IMMUNITY,  AND  RECOVERY. 

THE  tissues  of  the  animal  body  under  the  normal 
conditions  of  life  are,  as  we  have  seen,  unsuitable  for 
the  growth  of  the  great  majority  of  the  varieties  of 
bacteria.  Indeed,  only  a  very  small  number  of  the 
parasitic  bacteria  find  the  conditions  really  satisfactory, 
and  even  these  must  find  a  point  of  entrance  into  the 
body. 

In  seeking  to  account  for  the  difficulty  which  to  a 
greater  or  less  extent  all  bacteria  find  to  growing  in 
the  tissues  of  the  living  body,  we  cannot  find  it  either 
in  the  insufficient  or  excessive  concentration  of  the 
nutritive  substances,  nor  in  the  temperature,  nor  in  the 
reaction;  for  although  some  of  these  conditions  may  be 
unsuitable  for  some  bacteria,  they  are  all  suitable  for 
many,  and  thus  cannot  constitute  the  fundamental  ex- 
planation of  either  natural  or  acquired  immunity.  A 
possible  ground,  for  the  inability  of  the  bacteria  to 
invade  living  tissues,  might  be  thought  to  be  found  in 
the  fact  that  the  nutritious  material  in  the  living  cells 
is  in  a  form  which  the  bacteria  cannot  readily  assimi- 
late; but,  if  this  be  true  to  a  certain  extent,  it  does  not 
adequately  explain  why  the  bacteria  do  not  develop  in 
the  nutritious  fluids,  so  abundant  about  and  in  the  body 
tissues,  nor  does  it  account  for  acquired  immunity.  We 
are  thus  driven  to  the  conclusion  that  the. body  fluids 
themselves  contain  substances  which  are  directly  dele- 


INFECTION,  IMMUNITY,  AND  RECOVERY.     H9 

terious  to  the  bacteria.  As  to  the  origin  of  these 
substances,  we  may  conceive  that  they  may  be  either 
regularly  produced  by  the  body  cells,  or  by  the  fluids, 
or  by  both,  or  that  they  may  only  be  produced  or  at 
least  increased  when  bacteria  invade  the  body.  When 
formed  they  may  remain  unaltered  in  the  fluids  or  be 
quickly  eliminated  or  destroyed.  It  is  probable  that 
more  than  one  of  these  suppositions  is  actually  true. 

The  bactericidal  effect  upon  most  bacteria  of  the  body 
fluids,  noted  by  Nuttall  in  1888,  is  now  undisputed, 
and  is  shown  by  the  fact  that  bacteria  when  injected 
into  the  blood  usually  soon  die,  and  this  destruction 
may  be  so  rapid  that  in  a  few  hours  none  remain  alive. 
Even  when  bacteria  survive  and  produce  infection 
there  is  for  a  time  a  decrease  in  the  number  living, 
but  this  is  soon  followed  by  a  progressive  increase. 
This  fact  can  be  observed  not  only  by  injecting  bac- 
teria into  the  blood  and  peritoneal  cavity,  but  also  when 
the  bacteria  are  placed  in  the  animal  body  after  being 
enclosed  in  capsules.  The  bacteria  are  killed  even  if 
they  have  previously  grown  outside  the  body  in  blood- 
serum.  Bacteria  have  also  been  injected  into  a  vein 
carefully  ligated  above  and  below,  and  here,  without 
coagulation,  the  blood  exerts  bactericidal  properties. 
The  general  germicidal  effect  of  the  blood-serum  can 
also  be  watched  outside  of  the  body.  Here  mixed 
with  it  some  species  of  bacteria  die  quickly,  some 
slowly,  and  some  lose  only  a  portion  of  their  number, 
those  remaining  alive  after  a  time  rapidly  increasing. 
The  number  of  bacteria  introduced  is  of  great  impor- 
tance, for  the  serum  with  its  contained  substances  seems 
capable  of  destroying  only  a  certain  number,  and  after 
that  loses  its  bactericidal  properties. 


120  BACTERIOLOGY. 

If  the  bactericidal  effect  of  the  serum  outside  the 
body  always  went  hand-in-hand  with  the  immunity  of 
the  individual  from  which  it  was  taken,  the  immediate 
cause  of  immunity  would  be  solved  and  our  search  be 
directed  to  find  the  source  and  nature  of  these  germi- 
cidal  substances;  but  this  is  not  wholly  the  case,  for 
while  in  many  instances  it  is  so,  in  others  it  is  as  un- 
doubtedly not  true.  We  must,  therefore,  add  to  the 
serum  the  activity  of  the  cells,  which  produce  constantly 
the  substances  which  are  partly  given  up  to  the  blood 
and  fluids  of  the  body  and  partly  retained  in  their  own 
bodies.  This  deleterious  action  of  the  blood  in  bac- 
teria can  be  increased  by  infection.  Some  good  ob- 
servers have  found  that  blood  in  animals  naturally 
immune  to  certain  parasitic  bacteria,  which  had  little 
or  110  bactericidal  effect,  became  possessed  of  it  after  a 
moderate  infection;  this  seeming  to  indicate  a  protective 
effort  of  the  body  cells  to  withstand  bacterial  invasion. 

Concerning  the  nature  of  these  non-specific  protective 
substances,  named  alexines  by  Buchner,  we  have  as  yet 
little  positive  knowledge,  but  certain  properties  of  them 
are  known.  They  are  largely  precipitated  by  a  40  per 
cent,  solution  of  sodium  sulphate,  but  not  by  alcohol. 
These  substances  would  seem  to  belong  to  the  so-called 
living  proteids,  and  resemble  certain  of  the  globulins 
in  their  properties,  but  they  are  evidently  extremely 
complex  in  their  nature.  Many  of  them  become  inert 
on  standing  for  several  months,  even  at  low  tempera- 
tures, and  after  a  few  weeks  at  blood-heat.  A  tem- 
perature above  62°  to  70°  C.  soon  totally  destroys 
them.  Freezing  does  not  affect  them.  A  bactericidal 
serum  affects  in  a  deleterious  manner  the  red  blood- 
cells  of  a  different  species  of  animals. 


INFECTION,  IMMUNITY,  AND  RECOVERY.     121 

Their  source  must  apparently  be  attributed  to  the  cells, 
but  probably  certain  cells  only  produce  them.  The  red 
blood-cells,  for  instance,  seem  rather  to  destroy  than  to 
increase  them.  The  nuclein  derived  from  the  cells,  al- 
though it  has  a  general  bactericidal  action,  and  may  enter 
into  the  alexines,  yet  as  it  has  different  properties  it  can- 
not itself  be  one  of  these  bodies.  The  cells  which  have 
abundant  nuclear  substance,  such  as  the  leucocytes  and 
lymph-cells,  seem  especially  to  be  a  source  of  the  alex- 
ines. Buchner  and  others  have  found  that  through 
the  irritation  of  bacterial  filtrates  the  leucocytes  were 
attracted  in  great  numbers  to  the  region  of  injection, 
and  that  the  fluid  here,  which  was  rich  in  leucocytes, 
was  more  bactericidal  than  that  of  the  blood-serum 
elsewhere.  The  same  fluid  acted  also  more  perfectly 
when  it  contained  numbers  of  leucocytes  than  when 
they  were  filtered  off.  Substances  similar  to  the  alex- 
ines are  apparently  derived  from  the  leucocytes,  and 
their  attraction  to  the  injected  area  gives  to  that  loca- 
tion greater  protective  power.  Some  claim  to  have 
demonstrated  that  along  with  increased  leucocytosis 
there  is  a  general  increase  in  the  alexines  in  the  blood, 
still  it  has  not  yet  been  positively  established  that  the 
alexines  are  derived  solely  from  the  leucocytes,  nor 
from  all  leucocytes,  and  a  mere  increase  in  them  does 
not  always  mean  an  increase  in  alexines.  The  attrac- 
tion between  the  leucocytes  and  the  bacteria  is  due  to 
the  chemical  attraction  between  them  and  the  bacterial 
body  substance  and  its  poisons.  Some  chemical  sub- 
stances not  derived  from  bacteria  have  this  quality 
also,  called  positive  chemotaxis,  while  others  repel  the 
leucocytes — negative  chemotaxis.  The  original  theory 
of  Metchnikoff,  that  the  leucocytes  were  the  only  actual 


/ 


122  BACTERIOLOGY. 

protective  bodies  which  warded  off  disease,  and  that 
they  did  this  by  attacking  the  bacteria,  was  founded 
on  the  observation  that  certain  of  the  white  cells  pos- 
sessed the  power  of  taking  up  into  themselves  patho- 
genic bacteria,  and  that  they  were  there  destroyed.  It 
was  later  observed  that  these  cells  had  the  property  of 
taking  from  the  blood  many  lifeless  foreign  elements, 
thereby  keeping  the  blood-channels  free  of  foreign  par- 
ticles. The  question  thereby  arose  as  to  whether  these 
cells  engulfed  and  then  killed  the  bacteria,  or  whether 
perhaps  other  influences  killed  or  injured  them  before 
the  cells  took  them  up.  The  theory  then  became  some- 
what modified,  more  knowledge  was  obtained,  and  it  is 
now  believed  that  the  bacterial  substances  attract  the 
cells,  and  that  when  these  cells  are  brought  together  the 
general,  and  perhaps  the  specific,  bactericidal  property 
of  the  blood  in  their  neighborhood  is  thereby  increased. 
The  death  of  the  bacteria  liberates  this  positive  chemo- 
taxic  substance,  and  the  disintegration  of  the  white 
blood-cells  gives  rise  to  the  bactericidal  bodies.  Thus 
we  find  that  phagocytosis  is  most  marked  when  the  dis- 
ease is  on  the  decline  or  the  infection  mild,  but  that  in 
rapidly  increasing  progressive  infection  it  is  absent. 
This  would  seem  to  indicate  that  the  course  of  the  in- 
fection is  often  already  determined  before  the  leucocytes 
become  massed  at  the  point  of  its  entrance.  The  first 
determining  influence  is  given  by  the  condition  of  the 
tissues  and  the  bactericidal  substances  contained  in 
them,  and  then,  later,  in  cases  where  the  infection  is 
checked,  comes  the  additional  bactericidal  substance 
given  off  by  the  attracted  leucocytes.  In  so  far  as  the 
tissues  themselves  are  unsuitable  for  the  development 
of  bacteria  they  are  sufficient  to  ward  off  infection,  but 


INFECTION,  IMMUNITY,  AND  RECOVERY.     123 

in  proportion  as  they  are  incapable  of  doing  this  they 
are  assisted  by  the  substances  contained  in  the  leuco- 
cytes. If  the  tissues  are  wholly  adapted  .for  the 
growth  of  the  bacteria,  neither  they  nor  the  leuco- 
cytes, nor  both  combined,  can  furnish  sufficient  pro- 
tective substances  to  prevent  the  bacterial  increase. 
The  entrance  of  bacteria  into  the  leucocytes,  which  is 
not  infrequent,  may  mean  their  destruction;  but,  on 
the  other  hand,  the  bacteria  may  increase  in  the  white 
blood-cells  and  destroy  them,  and  they  may  be  killed 
without  entering  the  cells.  The  simple  absorption  by 
the  cells  is  not  necessarily  a  destructive  process.  No 
explanation  can  as  yet  be  given  of  natural  immunity 
to  bacterial  poisons,  except  that  it  may  be  connected 
with  some  general  property  of  the  tissue.  There  is  far 
less  variation  among  different  species  in  their  resistance 
to  the  bacterial  poisons  than  in  their  suitability  for  the 
growth  of  the  living  bacteria  which  produce  them 
Possibly  certain  organs,  such  as  those  which  are  rich 
in  nuclei n — for  example,  the  lymph-glands,  the  liver, 
etc. — may  have  some  destructive  power  with  regard  to 
poisons.  The  nature  of  the  cell  substance  is  known 
to  have  much  to  do  with  its  relations  to  certain  poisons. 
Thus  the  tetanus  poison  acts  chiefly  on  the  nerve  cells 
and  leaves  the  others  almost  or  altogether  unaffected. 

By  what  means  are  virulent  bacteria  enabled  to  in- 
crease in  the  body, notwithstanding  its  protective  powers, 
when  non-virulent  organisms  of  the  same  species  are 
incapable  of  so  doing?  This  is  but  little  understood, 
but  experiment  shows  in  the  first  place  that  both  viru- 
lent and  non-virulent  forms  are  equally  resistant  to 
general  destructive  agencies;  and,  second,  that  the  bac- 
teria are  capable  of  producing  substances  (lysines)  which 


124  BACTERIOLOGY. 

neutralize  in  some  way  the  protective  substances  (alex- 
ines).  The  virulence  of  bacteria  would,  therefore,  de- 
pend partly  upon  their  ability  to  produce  these  lysines, 
which  act  perhaps  as  the  ferments  upon  the  alexines,  or 
perhaps  combine  with  them.  That  bacteria  under  certain 
conditions  form  specific  poisons,  and  under  others,  even 
when  they  grow  luxuriantly,  do  not,  is  clearly  shown  by 
our  experiments  on  the  production  of  diphtheria  toxin. 
Here,  as  previously  stated,  it  was  found  that  when 
the  bouillon  was  either  a  little  too  alkaline  or  too  acid, 
though  the  bacilli  grew  rapidly,  they  did  not  produce 
specific  toxins.  By  growing  the  bacilli  for  a  time  in 
such  bouillon  they  eventually  became  able  to  develop 
toxin  in  a  soil  in  which  they  previously  failed  to  do  so. 
Similar  cultivation  in  the  body  may  be  assumed  to 
increase  their  ability  to  produce  specific  poison  after  a 
while  under  what  would  at  first  be  adverse  conditions. 

With  regard  to  the  increase  and  decrease  of  general, 
and  perhaps  also  of  specific  immunity,  we  have  reason 
to  believe  that  as  the  protective  substances  are  produced 
by  the  living  cells,  anything  which  lowers  the  general 
vitality  must  lessen  the  vitality  of  the  cells,  and  thus 
their  ability  to  produce  protective  substances  in  the 
amount  possible  in  a  normal  condition.  The  attraction 
of  leucocytes  to  any  point  by  some  new  infection  might 
increase  the  germicidal  action  of  the  tissues,  and  so  in- 
fluence the  first  infection. 

Specific  Immunity.  The  following  theories  have  been 
advanced  concerning  the  nature  of  specific  immunity: 
The  theory  that  a  second  infection  is  impossible  because 
the  first  used  up  substances  which  were  necessary  to  the 
growth  of  the  bacteria  is  untenable  for  many  reasons. 
Thus  it  can  be  demonstrated  that  the  injection  of  a 


INFECTION,  IMMUNITY,  AND  RECOVERY.     125 

small  amount  of  specific  serum,  which  robs  the  tissues 
of  nothing,  produces  the  same  immunity.  Again,  the 
injection  into  the  body  of  a  sufficient  number  of  patho- 
genic bacteria  gives  rise  to  an  infection  in  all  cases. 

The  theory  of  Metchnikoff,  that  the  leucocytes  or 
wandering  cells  of  the  body,  after  an  infection  with  a 
certain  variety  of  bacteria,  become  influenced  in  some 
way,  so  that  they  attack  especially  that  form  of  infection 
again  and  destroy  the  bacteria  (phagocytosis),  can  no 
longer  be  considered  as  more  than  a  very  partial  ex- 
planation, and  can  only  be  accepted  by  assuming  as 
proven  a  number  of  hypotheses. 

The  retention  theory  of  Wernich  and  Chaveau,  some- 
what modified,  has  much  to  support  it.  The  blood- 
serum  of  animals  recovering  from  an  infection  was  found 
to  have  changed  chemically  to  such  an  extent  as  to  be 
capable  of  being  demonstrated  experimentally,  and 
these  changes  were  shown  to  persist  for  a  number 
of  weeks  or  months  or  even  years.  Similarly  the  serum 
of  immunized  animals  retains  for  a  long  time  its  immu- 
nizing substances  We  are,  therefore,  compelled  to  ac- 
cept the  fact,  that  when  an  infection  is  passed  through 
there  are  more  or  less  protective  chemical  substances 
left  in  the  blood,  which  remain  there  for  a  considerable 
time.  Kruse  believes  that  these  substances  have  the 
power  of  neutralizing  the  bacterial  poisons  which  are 
given  off  by  the  bacteria  upon  their  entrance  into  the 
body,  and  of  thus  robbing  them  of  their  deleterious 
effects  on  the  alexines;  the  body  fluids  in  this  way 
remaining  unsuitable  soil  for  the  growth  of  the  bac- 
teria, the  alexiues  being  bactericidal.  If  only  a  small 
amount  of  antilysines  are  present  some  of  the  alexines 
are  destroyed  and  the  bacteria  are  not  all  killed  or 


126  BACTERIOLOGY. 

weakened.  Those  remaining  active  are  then  further 
acted  upon  by  the  alexines  in  the  tissues  and  by  the 
substances  given  off  by  the  leucocytes.  If  these  pro- 
tective substances  are  insufficient  the  infection  is  estab- 
lished. R.  Pfeiffer's  experiments  with  cholera  and 
typhoid  cultures  injected  into  the  peritoneal  cavity  of 
the  guinea-pig  along  with  specific  protective  serum 
showed  that  the  bacteria  were  altered  and  destroyed  by 
the  serum  within  a  few  minutes,  just  as  if  they  had 
been  non-virulent  bacteria,  and  this  without  the  assist- 
ance of  the  phagocytes.  In  this  case  non-virulent  bac- 
teria die  because  they  produce  no  ly sines  to  destroy  the 
alexines,  while  those  which  are  virulent  do  not  thrive, 
because  although  they  produce  lysines,  the  antilysines 
in  the  serum  destroy  them,  being  thus  acted  upon  by 
alexines  in  like  manner  to  the  non-virulent  bacteria. 

As  to  the  development  of  the  specific  protective  sub- 
stances, the  most  plausible  theory  seems  to  us  to  be  that 
they  are  formed  by  the  activity  of  the  cells  from  the 
bacterial  poisons,  the  lysiues.  These  substances  are 
stated  by  Pfeiffer  and  Marx  to  be  most  abundant  in  the 
spleen,  lymphatic  glands,  and  bone-marrow. 

Ehrlich  and  others  believe  antitoxin  to  be  a  portion 
of  the  substance  of  certain  cells,  which,  having  been 
stimulated  by  their  effort  to  replace  portions  of  their 
substances  destroyed  by  previous  doses  of  toxin,  have 
reproduced  it  in  excess.  This  cell  substance,  being 
free  in  the  fluids  of  the  body,  combines  with  the  toxin, 
and  thus  neutralizes  it.  But  it  is  difficult  by  this 
theory  to  explain  many  known  facts,  such  as  the  one 
that  a  fully  neutralized  mixture  of  toxin  and  antitoxin 
is  still  capable  of  producing  in  the  body  more  anti- 
toxin. Others  hold  that  the  antitoxins,  as  the  other 


INFECTION,  IMMUNITY,  AND  RECOVERY.     127 

protective  substances,  are  always  present  in  the  body, 
and  that  under  special  need,  as  when  bacterial  inva- 
sion takes  place,  they  are  thrown  out  by  the  cells  in 
larger  quantities,  and  this  is  especially  true  of  the 
special  substances  needed  for  existing  infection.  The 
antitoxin  then  acts  by  fortifying  the  cells  so  that  they 
are  enabled  to  resist  the  action  of  the  toxin.  In  favor 
of  this  view  is  the  fact  that  the  cells  of  certain  ani- 
mals are  undoubtedly  proof  against  these  toxins,  and 
yet  so  far  as  chemistry  in  its  present  development  can 
detect,  these  cells  are  the  same  as  similar  but  sensitive 
cells  in  other  animals.  Another  theory  is  that  the 
toxin,  in  some  way  in  the  body  fluids,  is  changed  into 
antitoxin.  This  is  made  slightly  plausible  by  the  fact 
that  by  the  action  of  electricity  there  have  been  obtained 
substances  from  toxins  which  are  slightly  antitoxic. 
The  practical  point  to  remember  is  that  whether  or  not 
the  theories  are  correct,  there  is  no  doubt  that  the  pro- 
tective substances  exist. 


CHAPTER  VII. 

INFECTION. 

THE  spread  of  infection  is  influenced  by:  1.  The 
number  of  species  of  animals  subject  to  infection. 

Many  human  infectious  diseases  do  not  occur  in  ani- 
mals, and  many  animal  infections  are  not  found  in  man. 
Thus,  so  far  as  we  know,  gonorrhoea,  syphilis,  measles, 
smallpox,  typhoid  fever,  etc.,  do  not  occur  in  animals 
under  ordinary  conditions;  while  tuberculosis,  anthrax, 
glanders,  hydrophobia,  and  some  other  diseases  are 
common  to  both  man  and  animals. 

2.  The  quantity  of  the  infectious  material  thrown  off 
from  the  body  and  the  prevalence  of  the  disease. 

In  diphtheria,  typhoid  fever,  cholera,  pulmonary 
tuberculosis,  septic  endometritis,  influenza,  and  gonor- 
rhoea enormous  numbers  of  infectious  bacteria  are  cast 
off  through  the  discharges  from  the  mouth,  intestines, 
and  genito  urinary  secretions,  causing  great  danger  of 
infection.  On  the  other  hand,  in  tubercular  perito- 
nitis, cerebro- spinal  meningitis,  septic  endocarditis, 
gonorrhoeal  rheumatism  and  the  like,  there  is  little  or 
no  danger  of  infecting  others,  as  few  or  no  bacteria  are 
cast  off. 

3.  The  resistance  of  the  infectious  bacteria  to  the 
deleterious  effects  of  drying,  light,  heat,  etc. 

In  this  case  the  presence  or  absence  of  spores  is  of 
the  greatest  importance.  The  spore-bearing  bacilli, 


INFECTION.  129 

such  as  tetanus,  anthrax,  etc.,  being  able  to  withstand 
destruction  for  a  long  time,  retain  their  power  of  pro- 
ducing infection  for  months  or  even  years  after  elimi- 
nation from  the  body.  The  bacteria  which  form  no 
spores  show  great  variation  in  their  resistance  to  out- 
side influences.  Some  of  these,  such  as  the  influenza 
bacilli  and  the  gonococci,  the  virus  of  syphilis  and 
hydrophobia,  are  extremely  sensitive;  the  pneumococci, 
cholera  spirilla,  glanders  bacilli,  etc.,  are  a  little  hardier; 
then  follow  the  diphtheria  bacilli,  and  after  them  the 
typhoid  and  tubercle  bacilli  and  the  staphylococci. 

4.  The  ability  or  the  lack  of  ability  to  grow  outside 
of  the  infected  tissues. 

Such  bacteria  as  the  pneumococcus,  tubercle,  influ- 
enza, and  lebrosy  bacilli  do  not  develop,  as  far  as  we 
know,  outside  of  the  body  under  ordinary  conditions. 
Others,  like  the  diphtheria  bacillus  and  the  strepto- 
coccus, may  under  certain  conditions,  as  in  milk  in 
warm  places,  develop  and  produce  infection.  Others, 
again,  such  as  the  streptococcus  and  staphylococcus, 
typhoid  and  anthrax  bacillus,  the  cholera  spirillum, 
and  some  anaerobics,  may  develop  under  peculiar  con- 
ditions existing  in  water  or  soil. 

While  for  the  pathogenic  bacteria,  as  a  rule,  the 
saprophytes  met  with  in  the  soil  and  water  are  antago- 
nistic, yet  in  some  cases — and  especially  is  this  true  of 
the  anaerobic  bacteria — they  are  helpful.  Such  bacilli 
as  tetanus  are  believed  to  require  the  associaton  of  an- 
aerobic bacteria  to  permit  of  their  development  in  the 
soil  in  the  presence  of  oxygen. 

A  large  number  of  the  infectious  bacteria  are  able  to 
develop  in  or  upon  some  portion  of  the  skin  or  mucous 
membrane,  either  after  or  before  disease,  and  without 

9 


130  BACTERIOLOGY. 

causing  infection.  As  complete  a  knowledge  of  these 
facts  as  possible  is  necessary  if  we  are  to  combat  the 
spread  of  infection.  In  the  superficial  layers  of  the 
epithelium  and  on  the  surface  of  the  skin  we  find  the 
different  pyogenic  cocci,  which  are  capable  of  infecting 
a  wounded  or  injured  part  or  causing  inflammation  in 
the  glands.  Acne,  the  pustules  in  smallpox,  the  pus 
on  a  burned  surface,  boils,  etc.,  all  come  from  the 
pyogenic  cocci.  In  surgical  cases  the  skin  has  to  be 
as  thoroughly  disinfected  as  possible,  to  prevent  the 
formation  of  stitch-hole  abscesses  and  wound- suppura- 
tion. 

In  the  secretion  of  the  mucous  membrane  covering 
the  pharynx  and  nasopharynx  there  is  always  an  abund- 
ance of  bacteria.  In  one  hundred  throats  examined  by 
the  writer  in  New  York  City,  streptococci  and  staphylo- 
cocci  could  be  found  in  over  90  per  cent.,  and  pneumo- 
cocci  were  very  frequently  discovered.  Many  other 
varieties  of  bacteria,  such  as  the  influenza  bacilli,  are 
probably  often  present  in  small  numbers.  In  those  con- 
stantly in  contact  with  cases  of  diphtheria,  and  in  those 
convalescent  from  diphtheria,  virulent  diphtheria  bacilli 
are  frequently  found  in  the  throat. 

After  exposure  to  cold  or  injury  of  any  kind,  owing 
to  the  presence  of  these  bacteria,  the  persons  harboring 
them  may  develop  tonsillitis,  tonsillar  abscess,  or  diph- 
theria; or  the  bacteria  may  invade  the  bronchial  mucous 
membrane  or  the  lungs.  The  diphtheria  bacilli,  and 
perhaps  other  bacteria,  transmitted  to  others  may  be- 
come the  source  of  infection  to  them,  though  the  person 
who  spreads  the  infection  may  remain  unaffected. 

The  stomach,  on  account  of  the  acidity  of  its  con- 
tents, is'Vomparatively  free  from  bacteria.  The  normal 


INFECTION.  131 

intestines,  on  the  other  hand,  contain  great  numbers  of 
bacteria.  Among  these  the  colon  bacillus  is  constantly 
present,  and  often  the  streptococcus  and  other  patho- 
genic bacteria.  After  typhoid  fever  the  bacilli  may 
remain  in  the  intestinal  contents  for  weeks  and  in  the 
bladder  and  gall-bladder  for  months.  The  bacteria 
swallowed  to  a  considerable  extent  escape  destruction 
in  the  stomach,  and  thus  appear  in  the  intestines.  Some 
good  observers  have  stated  that  bacteria  can  be  absorbed 
through  the  intestinal  wall  into  the  chyle  and  blood. 
When  the  intestinal  canal  is  injured,  or  its  circulation 
hindered  by  strangulation,  etc.,  the  bacillus  coli  and 
some  other  bacteria  may  penetrate  through  the  injured 
walls  and  cause  peritonitis  or  general  infection.  Under 
certain  conditions,  as  during  the  debility  due  to  hot 
weather,  the  bacteria  in  the  intestines  cause,  through 
their  products,  irritation,  and  in  children  even  serious 
intestinal  inflammation. 

The  kidneys,  bladder,  and  urethra  may  be  the  source 
of  infection  and  may  give  rise  to  disease  in  others. 
Long  after  an  acute  gonorrhnea  has  passed  gonococci 
may  remain  in  sufficient  numbers  to  cause  a  new  in- 
flammation or  produce  infection  in  others.  A  cystitis 
may  run  on  chronically  for  years,  and  then  suddenly 
become  acute  or  spread  infection  to  the  kidneys.  After 
typhoid  fever  the  urine  may  contain  abundant  typhoid 
bacilli  for  weeks  and  be  little  thought  of  as  a  source  of 
infection.  A  persistent  gonorrhoeal  vaginal  infection 
may  lead  to  a  gonorrhoeal  endometritis,  or  salpingitis,  or 
peritonitis,  under  suitable  conditions.  The  staphylococci 
in  the  skin  and  the  colon  bacilli  and  pyogenic  cocci  in 
the  faecal  discharges  may  also  be  carried  into  the  uterus 
and  produce  septic  infection. 


132  BACTERIOLOGY. 

Inherited  Infection  and  Susceptibility  to  or  Immunity 
from  Infection.  The  passage  of  bacteria  from  the 
mother's  blood  through  the  placenta  to  the  foetus  has 
been  demonstrated  for  numerous  bacteria,  among  the 
most  important  of  which  may  be  mentioned  the  pneu- 
mococcus,  streptococcus,  and  tubercle  bacillus.  The 
detection  of  the  tubercle  bacillus  by  Gartner  and  others 
under  these  circumstances  prevents  us  from  denying  the 
possibility  that  tuberculosis  developing  in  children  may 
have  been  due  to  infection  taking  place  before  birth. 
The  fact,  however,  that  calves  removed  from  tubercu- 
lous cattle  and  fed  on  milk  free  from  tubercle  bacilli  do 
not  develop  tuberculosis,  while  those  left  with  tubercu- 
lous cattle  become  tuberculous,  indicates  that  tubercu- 
losis in  man  also  is  usually,  at  least,  due  to  infection 
after  birth.  The  infection  from  spermatozoa  is  con- 
ceivably possible  in  tuberculosis  if  the  testicles  are 
affected;  the  same  may  be  said  of  syphilis;  but  except 
for  syphilis,  in  which  the  nature  of  the  infective  agent 
is  unknown,  we  believe  that  such  infection  is,  if  ever 
present,  extremely  rare. 

Natural  immunity  pertains  more  to  species  than  in- 
dividuals, and  such  immunity  is  handed  down  by  the 
parents  to  their  offspring.  If  the  immunity  of  one  or 
both  parents  has  been  acquired  by  them  during  their 
lifetime  previous  to  the  birth  of  the  offspring  the  im- 
munity conferred  is  slight  or  none  at  all.  This  is  espe- 
cially true  of  the  male  side.  In  the  case  of  the  female 
parent  another  factor  comes  into  play  after  the  fructi- 
fication of  the  ovum — viz.,  the  absorption  of  products 
from  the  fluids  of  the  mother,  for  the  placenta  is  no 
barrier  to  soluble  substances.  Thus,  sheep  which  have 
been  immunized  to  anthrax  have  moderately  immune 


INFECTION.  133 

young.  On  the  other  hand,  animals  vaccinated  with 
cowpox  have  not  been  found  to  have  immune  offspring. 
Toxins  injected  into  the  parents  apparently  do  not  pass 
the  placenta;  but  antitoxins  do,  giving  thus  a  slight 
transitory  passive  immunity.  A  slight  immunity  is 
also  given  by  immune  mothers  through  their  milk,  a 
small  amount  of  antitoxic  substance  being  absorbed. 


CHAPTER   VIII. 

THE  EFFECT  OF  LIGHT,  ELECTRICITY,  PRESSURE, 
AGITATION,  DRYING,  AND  ASSOCIATION  WITH  OTHER 
MICRO-ORGANISMS  UPON  BACTERIA. 

VERY  little  is  known  about  the  influence  of  electricity 
on  bacteria.  The  majority  of  the  observations  hereto- 
fore made  on  this  subject  would  seem  to  indicate  that 
there  is  no  direct  action  of  the  galvanic  current  on 
bacteria;  but  the  effect  of  heat  and  the  electrolytic  in- 
fluence on  the  culture  liquid  may  produce  changes  which 
finally  sterilize  it.  Gottstein  and  Spilker  have  recently 
made  experiments  with  an  induction  current  from  a 
dynamo  machine.  They  passed  the  current  through 
a  spiral  wire,  which  was  wrapped  around  a  test-tube 
of  glass  containing  the  micro-organisms  to  be  tested, 
suspended  in  water.  The  bacillus  prodigiosus,  sus- 
pended in  sterilized  distilled  water  and  contained  in 
test-tubes  having  a  capacity  of  250  c.c.,  was  subjected 
to  a  current  of  2.5  amperes  -{-  1.25  volts  for  twenty- 
four  hours.  The  temperature  did  not  go  above  30°  C. 
No  growth  occurred  when  the  organism  tested  was  sub- 
sequently planted  in  nutrient  gelatin.  It  was  found  that 
stronger  currents  were  effective  in  a  shorter  time,  but 
in  no  case  was  sterilization  effected  in  less  than  an  hour. 
These  experiments,  however,  have  not  been  confirmed. 

Meltzer  has  shown  that  while  slight  agitation  of 
cultures  of  bacteria  acts  favorably  on  their  develop- 


INFLUENCE  OF  LIGHT.  135 

merit,  the  vitality  of  bacteria  is  destroyed  by  protracted 
and  violent  shaking,  which  causes  a  molecular  disinte- 
gration of  the  cells. 

D'  Arson val  and  Charrin  submitted  a  culture  of  bacil- 
lus pyocyaneus  to  a  pressure  of  fifty  atmospheres  under 
carbonic  acid.  At  the  end  of  four  hours  cultures  could 
still  be  obtained,  but  the  bacillus  had  lost  its  power  of 
pigment  production.  A  few  colonies  were  developed 
after  six  hours7  exposure  to  this  pressure,  but  after 
twenty-four  hours  no  development  occurred. 

Influence  of  Light.  A  large  number — perhaps  the 
majority — of  bacteria  are  inhibited  in  growth  by  the 
action  of  diffuse  daylight,  still  more  by  that  of  direct 
sunlight,  and  when  the  action  is  prolonged  they  lose 
their  power  of  developing  when  later  placed  in  the  dark. 

In  order  to  test  the  susceptibility  of  bacteria  to 
light,  it  is  best,  according  to  Buchner,  to  suspend  a 
large  number  of  bacteria  in  nutrient  gelatin  or  agar 
and  pour  the  media  while  still  fluid  in  Petri  dishes, 
upon  which  has  been  pasted  a  strip  of  black  paper  on 
the  side  upon  which  the  light  is  to  act.  The  action  of 
heat  may  be  shut  off  by  allowing  the  ray  of  light  to 
pass  first  through  a  layer  of  water  or  alum  of  several 
centimetres'  thickness.  After  the  plates  have  been 
exposed  to  the  light  for  one-half,  one,  one  and  a  half, 
two  hours,  etc.,  they  are  taken  into  a  dark  room  and 
allowed  to  stand  at  20°  or  35°  C.,  a  sufficient  length 
of  time  to  allow  of  growth,  and  then  examined,  to  see 
whether  there  are  colonies  anywhere  except  on  the  spot 
covered  by  the  paper;  when  the  colonies  exposed  to  the 
light  have  been  completely  destroyed  there  is  a  sharply 
defined  region  of  the  shape  of  the  paper  strip  crowded 
with  colonies  lying  in  a  clear  sterile  field. 


136  BACTERIOLOGY. 

Dieudonne,  in  experiments  upon  the  bacillus  pro- 
digiosus,  found  that  direct  sunlight  in  March,  July,  and 
August  killed  these  bacilli  in  one  and  a  half  hours;  in 
November  in  two  and  a  half  hours.  Diffuse  daylight 
in  March  and  July  restrained  development  after  three 
and  a  half  hours7  exposure  (in  November  four  and  a 
half  hours),  and  completely  destroyed  vitality  in  from 
five  to  six  hours.  Electric  arc-light  inhibited  growth 
in  five  hours  and  destroyed  vitality  in  eight  hours. 
Incandescent  light  inhibited  growth  in  from  seven  to 
eight  hours  and  killed  in  eleven  hours.  Similar  results 
have  been  obtained  with  B.  coli,  B.  typhosus,  and  B. 
anthracis.  According  to  Koch,  the  tubercle  bacillus  is 
killed  by  the  action  of  direct  sunlight  in  a  time  vary- 
ing from  a  few  minutes  to  several  hours,  depending 
upon  the  thickness  of  the  layer  exposed  and  the  season 
of  the  year.  Diffuse  daylight  also  had  the  same  effect, 
although  a  considerably  longer  time  of  exposure  was 
required — when  placed  close  to  a  window,  from  five  to 
seven  days. 

Only  the  ultraviolet,  violet,  and  blue  rays  of  the 
spectrum  seem  to  possess  bactericidal  action;  green 
light  is  very  much  less  so,  red  and  yellow  light  not  at 
all.  The  action  of  light  is  apparently  assisted  by  the 
admission  of  air;  anaerobic  species,  like  the  tetanus 
bacillus,  and  facultative  anaerobic  species,  such  as  the 
colon  bacillus,  are  able  to  withstand  quite  well  the 
action  of  sunlight  in  the  absence  of  oxygen,  the  B.  coli 
intense  direct  sunlight  for  four  hours. 

According  to  Kichardson  and  Dieudonne,  the  mech- 
anism of  the  action  of  light  may  be  at  least  partially 
explained  by  the  fact  that  in  agar  plates  exposed  to 
light  for  a  short  time  (even  after  ten  minutes7  exposure 


INFLUENCE  ONE  SPECIES  UPON  ANOTHER.     137 

to  direct  sunlight)  hydrogen  peroxide  (H2O2)  is  formed. 
This  is  demonstrated  by  exposing  an  agar  plate  half- 
covered  with  black  paper,  upon  which  a  weak  solution 
of  iodide  of  starch  is  poured,  and  over  this  again  a 
dilute  solution  of  sulphate  of  iron;  the  side  exposed 
to  the  light  turns  blue-black.  In  gases  containing  no 
oxygen,  hydrogen  peroxide  is  not  produced,  and  the 
light  has  no  injurious  effect.  Access  of  oxygen  also 
explains  the  effect  which  light  produces  on  culture 
media  which  have  been  exposed  to  the  action  of  sun- 
light, as  standing  in  the  sun  for  a  time,  when  after- 
ward used  for  inoculation.  The  bacteria  subsequently 
introduced  into  such  media  grow  badly — far  worse  than 
in  fresh  culture  media  which  are  kept  in  the  shade. 

Influence  of  One  Species  upon  the  Growth  of  Another. 
While  it  is  the  custom  of  bacteriologists  to  have  pure 
cultures  to  work  with,  we  should  never  forget  that  in 
nature  bacteria  often  occur  in  mixed  cultures.  If  we 
examine  water,  milk,  or  the  contents  of  the  intestines 
of  either  sick  or  healthy  persons  we  shall  always  find 
several  species  of  bacteria  occurring  together.  This 
admixture  may,  perhaps,  seem  to  us  at  first  merely 
accidental,  but  on  further  investigation  it  will  appear 
that  also  in  the  department  of  bacteriology  there  exist 
synergists  and  antagonists,  or  at  least  bacteria  which 
assist  or  oppose  one  another  mutually  or  one-sidedly. 
Nencki  speaks  of  symbiosis  and  enantobiosix. 

Gasse"  has  demonstrated  experimentally  the  existence 
of  antagonisms  by  inoculating  gelatin  streak  cultures  of 
various  bacteria;  it  is  found  that  many  species  will  not 
grow  at  all  or  only  sparingly  when  in  close  proximity 
to  some  other  species.  This  antagonism,  however,  is 
often  only  one-sided  in  character;  for  instance,  the 


138  BACTERIOLOGY. 

bacillus  fluorescens  putidus  grows  well  when  inoculated 
between  streaks  of  staphylococcus,  but  the  latter  micro- 
coccus  will  not  grow  at  all  when  inoculated  between 
cultures  of  the  bacillus  putidus,  the  growth  of  the 
staphylococcus  remaining  scanty  when  the  two  species 
are  inoculated  simultaneously.  Again,  when  gelatin 
or  agar  plates  are  made  from  two  different  species  of 
bacteria  inoculated  into  the  same  tubes  while  liquid  it 
may  be  observed  that  only  one  of  the  two  grows.  A 
third  method  of  making  this  experiment  is  to  simulta- 
neously inoculate  the  same  liquid  medium  with  two 
species,  and  then  examine  them  later,  both  microscop- 
ically and  in  thin  plates;  not  infrequently  the  one  spe- 
cies may  take  precedence  of  the  other,  which  it  finally 
overcomes  entirely.  The  practical  application  of  this 
is  to  make  only  very  thin  plates  for  the  estimation  of 
the  number  of  bacteria  or  the  isolation  of  pure  cultures. 

Finally,  bacteria  may  oppose  one  another  as  antago- 
nists in  the  animal  body.  As  Emmerich  has  shown, 
animals  infected  with  anthrax  may  often  be  cured  by  a 
secondary  infection  with  the  streptococcus. 

The  symbiotic  or  co-operative  action  of  bacteria  is  of 
still  greater  importance,  of  which  the  following  examples 
may  be  given: 

1.  Some  bacteria  thrive  better  in  association  with 
other   species  than  alone.     Certain  anaerobic  species 
grow  even  with   the   admission   of   air  if  only  other 
aerobic  species  are  present  (tetanus). 

2.  Certain  chemical  effects,  as,  for  instance,  the  de- 
composition of  nitrates  to  gaseous  nitrogen,  cannot  be 
produced  by  many  bacteria  alone,  but  only  when  two 
are  associated. 

3.  In  like  manner  it  is  observed  that  in  a  series  of 


INFLUENCE  OF  NUTRITION  AND  MOISTURE.    139 

soil  bacteria  none  by  itself  may  be  pathogenic,  but 
when  inoculated  into  animals  in  certain  combinations 
produce  disease. 

4.  Slightly  pathogenic  species,  such  as  attenuated 
tetanus  bacilli,  for  example,  gain  in  virulence  when 
cultivated  together  with  the  proteus  vulgaris. 

Want  of  Nutrition  and  Water.  When  bacteria  which 
require  much  organic  food  for  their  development,  and 
these  include  most  of  the  pathogenic  species,  are  placed 
in  distilled  water  they  soon  die — that  is,  within  a  few 
days;  even  in  sterilized  well-water  their  life-duration 
does  not  usually  exceed  eight  to  fourteen  days,  and  they 
rarely  multiply.  Instances,  however,  of  much  more 
extended  life  under  certain  conditions  are  recorded. 
Want  of  water  affects  bacteria  in  different  ways. 
Upon  dried  culture  media  development  soon  ceases;  but 
in  media  dried  gradually  at  the  room-temperature 
(nutrient  agar,  gelatin,  potato)  they  live  often  for  a 
longtime,  even  when  there  are  no  spores  to  account  for 
it.  A  shrunken  residue  of  such  cultures  in  bouillon  has 
often  been  found,  after  a  year  or  more,  to  yield  living 
bacteria.  The  question  as  to  how  long  the  non-spore 
bearing  forms  are  capable  of  retaining  their  vitality 
when  dried  on  a  cover  glass  or  silk  threads  has  been 
variously  answered.  We  know  now  that  there  are 
many  factors  which  influence  the  retention  of  vitality. 
The  following  table  of  the  results  obtained  by  Sirena 
and  Alessi  gives  some  idea  of  the  extent  and  effect  of 
such  influences.  In  the  experiments  silk  threads  were 
saturated  with  bouillon  cultures  or  aqueous  suspensions 
of  the  bacteria,  and  some  then  enclosed  in  tubes  con- 
taining sulphuric  acid  or  calcium  chloride,  while  others 
were  left  exposed  to  various  outside  influences: 


140 


BACTERIOLOGY. 


Desiccation. 

With  sul- 
phuric 
acid, 
killed  at 
end  of 

With 
calcium 
chloride, 
killed  at 
end  of 

In  incu- 
bator at 
37°, 
killed  at 
end  of 

In  dry- 
room,  in 
shade, 
killed  at 
end  of 

In  moist 
room, 
killed  at 
end  of 

Cholera  spirilla     .    .    . 

1  day 

Iday 

1  day 

1  day 

12  days 

B.  of  fowl  cholera     .    . 

2  days 

1    " 

1    " 

5  days 

59    " 

B.  typhosus  

41    " 

1    " 

18  days 

64    " 

68    " 

B.  mallei   

35    " 

44  days 

31    " 

Diplococ.  pneumonias  . 

114    " 

31    " 

131    " 

164    " 

192     " 

The  spirillum  of  Asiatic  cholera  is  notorious  for  its 
slight  resistance  to  desiccation ;  according  to  the  ex- 
haustive investigations  of  Koch  and  the  above-men- 
tioned observers,  its  life-duration  is  only  from  one  to 
five  hours,  depending  upon  the  method  of  desiccation 
employed. 

The  results  of  all  investigators,  however,  would  seem 
to  indicate  that  the  greatest  possible  care  must  be  exer- 
cised in  desiccation  experiments  to  come  to  any  positive 
conclusions;  but  recently  most  astonishing  results  have 
been  obtained  with  regard  to  many  species  usually  sup- 
posed to  be  particularly  sensitive  to  desiccation,  showing 
that  under  certain  conditions  they  may  retain  their 
vitality  in  a  dry  state  for  a  very  long  time.  Thus, 
Koch  found  that  cholera  spirilla  lived  only  a  few  hours 
when  dry;  Kitasato  determined  their  life-duration  at 
fourteen  days  at  most;  while  Berckholz  and  various 
French  observers  have  found  that  they  may,  under 
favorable  conditions,  live  150  to  200  days.  The  vary- 
ing results  sometimes  reported  by  different  observers  in 
such  experiments  may  be  explained  by  the  fact  that  the 
conditions  under  which  they  were  made  were  different, 
depending  upon  the  desiccator  used,  the  medium  upon 


EFFECT  OF  OXYGEN.  141 

which  the  cultures  were  grown  and  the  use  of  silk 
threads  or  cover-glasses.  In  all  these  experiments,  of 
course,  it  should  be  previously  determined  that  in 
spore-bearing  species  there  are  no  spores  present. 

Behavior  Toward  Oxygen  and  Other  Gases.  As  already 
noted  under  the  nutritious  substances  required  by  bac- 
teria, it  is  customary  to  divide  bacteria  into  three  classes, 
according  to  their  behavior  toward  oxygen. 

1 .  Aerobic  Bacteria.     Growth  only  in  the  presence  of 
oxygen;  the  slightest  restriction  of  air  inhibits  devel- 
opment.    Spore-formation  especially  requires  the  free 
admission  of  air. 

2.  Anaerobic  Bacteria.     Growth  and  spore-formation 
only  in  the  total  exclusion  of  oxygen.    Among  this  class 
of  bacteria  are  the  bacillus  of  malignant  oedema,  the 
tetanus  bacillus,  the  bacillus  of  symptomatic  anthrax, 
and  many  soil  bacteria.     Exposed  to  the  action  of  oxy- 
gen, the  vegetative  forms  of  these  bacteria  are  readily 
destroyed;  these  spores,  on  the  contrary,  are  very  re- 
sistant.   Anaerobic  bacteria  being  deprived  of  oxygen — 
the  chief  source  of  energy  supplied  to  the  aerobic  spe- 
cies, by  which  they  oxidize  the  nutritive  substances  in 
the  culture  media — they  are  dependent  for  their  nutri- 
tion   upon   decomposable    substances,    such   as   grape- 
sugar,  which  on  separating  into  two  smaller  molecules 
— alcohol  and  carbonic  acid — give  out  energy  or  heat. 
Anaerobic  bacteria,  therefore,  require  for  their  cultiva- 
tion, as  a  rule,  media  containing  1  to  2  per  cent,  of 
glucose  or  some  equivalent. 

3.  Facultative  Aerobic  and  Facultative  Anaerobic  Bac- 
teria.   The  greater  number  of  aerobic  bacteria,  including 
most  of  the  pathogenic  species,  are  capable  of  withstand- 
ing, without  being  seriously  affected,  some  restriction 


142  BACTERIOLOGY. 

in  the  amount  of  oxygen  admitted,  and  many,  indeed, 
grow  equally  luxuriantly  in  the  partial  exclusion  of 
oxygen.  Life  in  the  animal  body,  for  example,  as  in 
the  intestines,  necessitates  existence  with  diminished 
supply  of  oxygen.  Pigment  formation  almost  always 
ceases  with  the  exclusion  of  oxygen,  but  poisonous 
products  of  decomposition  are  more  abundantly  pro- 
duced (Hueppe). 

It  is  important  to  note  that,  according  to  recent  in- 
vestigations, it  has  been  shown  that  the  aerobic  devel- 
opment of  the  anaerobes  may  be  facilitated  by  the  pres- 
ence of  living  or  dead  aerobes. 

It  has  also  been  observed  not  infrequently  that  certain 
species  which  on  their  isolation  at  first  showed  more  or 
less  anaerobic  development — that  is,  a  preference  to  grow 
in  the  depth  of  an  agar  stick  culture,  for  instance — 
after  a  while  seem  to  become  strict  aerobes,  growing 
only  on  the  surface  of  the  medium.  This  observation 
proves  that  the  simple  fact  of  an  organism  showing 
aerobic  for  anaerobic  growth  is  not  sufficient  for  its 
separation  into  a  distinct  species. 

While  all  facultative  as  well  as  strict  anaerobes  grow 
well  in  nitrogen  and  hydrogen,  they  behave  very  differ- 
ently toward  carbonic  acid  gas.  A  large  number  of 
these  species  do  not  grow  at  all,  being  completely  in- 
hibited in  their  development  until  oxygen  is  again 
admitted — for  example,  B.  anthracis  and  B.  subtilis 
and  other  allied  species.  It  has  been  found  in  some 
species,  as  glanders  and  cholera,  that  the  majority  of 
the  organisms  are  quickly  killed  by  CO2,  while  a  few 
offer  a  great  resistance,  rendering  impossible  complete 
sterilization  by  means  of  this  gas.  Another  group, 
again — viz.,  streptococcus  and  staphylococcus — exhibits 


EFFECT  OF  CARBONIC  ACID  GAS.  143 

a  scanty  growth;  while  a  third  group,  like  the  B. 
typhosus  and  B.  prodigiosus,  are  not  at  all  affected, 
growing  equally  well  as  in  the  presence  of  oxygen, 
and  the  liquefaction,  even  of  gelatin,  not  being  inter- 
fered with;  only,  on  account  of  the  lack  of  oxygen, 
there  is  no  p:gment  formation.  Finally,  a  mixture  of 
one-fourth  air  to  three-fourths  carbonic  acid  gas  seems 
to  have  no  injurious  effect  on  bacteria  which  cannot 
grow  in  an  atmosphere  of  pure  CO2. 

Sulphuretted  hydrogen  in  large  quantity  is  a  strong 
bacterial  poison,  and  even  in  small  amount  kills  some 
bacteria. 


CHAPTER   IX. 

EFFECT   OF    TEMPERATURE    UPON    BACTERIA. 

IN  judging  the  effect  on  bacteria  of  different  agents 
we  have  first  to  note  the  important  fact  that  different 
species  of  bacteria  are  differently  influenced  by  the  same 
substances.  Some  bacteria  thrive  under  conditions 
which  would  destroy  others,  and  they  vary  among 
themselves  in  their  powers  of  resistance  to  influences 
which  are  deleterious  to  all. 

Further,  any  species  of  bacteria  will  resist  better 
when  under  favorable  conditions  than  under  unfavor- 
able ones.  Bacteria  also  in  recent  cultures  withstand 
injury  better  than  those  in  old  cultures,  so  long  as  they 
have  not  entered  into  the  spore  form.  AccorcKng  to 
the  amount  of  injury  they  have  suffered,  bacteria  may 
be  only  lamed  in  some  of  their  functions  or  they  may 
be  totally  destroyed.  Their  loss  of  function  may  be 
only  temporary  or  permanent. 

Every  bacterial  species  makes  certain  demands  on 
the  temperature  of  its  culture  media.  Vegetative  life 
is  possible  within  the  limits  of  0°  and  70°  C.  There 
are  some  species,  however,  which  grow  at  the  lower  and 
others  at  the  upper  limit  of  these  temperatures.  The 
maximum  and  minimum  temperature  for  each  indi- 
vidual species  lies  about  30°  C.  apart.  Bacteria  have 
been  classified  according  to  the  temperatures  at  which 
they  develop,  as  follows  : 


EFFECT  OF  TEMPERATURE  UPON  BACTERIA.    145 

Psychrophilic  Bacteria.  Minimum  at  0°  C.,  optimum 
at  15°  to  20°  C.,  maximum  at  about  30°  C.  To  this 
class  belong  the  water  bacteria,  such  as  the  phosphor- 
escent bacteria  in  sea-water. 

Mesophilic  Bacteria.  Minimum  at  10°  to  15°  C., 
optimum  at  37°  C.,  maximum  at  about  45°  C.  To 
this  class  belong  all  pathogenic  bacteria,  the  conditions 
for  their  pathogenic  action  in  man  requiring  acclima- 
tization to  the  temperature  of  the  body. 

Thermophilic  Bacteria.  Minimum  at  40°  to  49°  C., 
optimum  at  50°  to  55°  C.,  maximum  at  60°  to  70°  C. 
This  class  includes  many  soil  bacteria  and  almost  ex- 
clusively spore-bearing  bacilli.  According  to  Globig, 
there  are  about  thirty  species  of  bacteria  capable  of  de- 
velopment at  60°  C.  and  a  few  at  70°  C.  M.iguel  has 
described  a  certain  bacillus  thermophillm,  which  thrives 
at  from  42°  to  72°  C.,  its  optimum  being  at  65°  to 
70°  C.,  and  found  in  cloacae,  the  contents  of  the  intes- 
tines, and  in  dirty  water.  Rabinowitsch  has  recently 
described  eight  thermophilic  facultative  anaerobic  spe- 
cies, all  spore-bearing,  non-motile  bacilli,  the  optimum 
temperature  of  which  is  from  60°  to  70°  C.,  though 
they  grow  slowly  at  from  34°  to  44°  C.,  and  best  on 
anaerobic  agar  cultures.  They  are  found  widely  dis- 
tributed in  the  feces. 

By  carefully  elevating  or  reducing  the  temperature 
Dieudonn6  has  succeeded  in  increasing  the  limits  within 
which  a  variety  of  bacteria  will  grow.  Thus,  anthrax 
was  gradually  made  to  accommodate  itself  to  a  tem- 
perature of  42°  C.,  and  pigeons,  which  are  compara- 
tively immune  to  anthrax,  on  account  of  their  high 
body  temperature  (42°  C.),  when  inoculated  with  this 
anthrax  succumbed  to  the  infection.  DieudonnS  also 

10 


146  BACTERIOLOGY. 

gradually  acclimated  anthrax  to  a  temperature  of 
12°  C.,  when  it  killed  frogs  kept  at  12°  C.  We  have 
cultivated  a  very  virulent  diphtheria  bacillus,  so  that 
it  will  grow  at  43°  C.  and  produce  strong  toxin. 

Bacterial  growth  is  retarded  by  temperatures  only  a 
little  below  the  minimum  of  the  species  in  question; 
but  they  are  not  otherwise  injured.  Indeed,  it  is  the 
usual  custom  in  laboratories  to  preserve  bacteria 
which  die  readily  (such  as  streptococci)  by  keeping 
them  in  the  refrigerator  at  about  4°  to  6°  C ,  after  cul- 
tivation for  two  days  at  20°  C.,  as  a  means  for  retain- 
ing their  vitality  without  repeated  transplantation. 
Temperatures  even  far  under*  0°  C.  are  only  slowly 
injurious  to  bacteria,  different  species  being  affected 
with  varying  rapidity.  Ordinarily,  low  temperatures, 
though  arresting  the  growth,  do  not  destroy  the  vitality 
of  bacteria.  This  has  been  demonstrated  by  numerous 
experiments  in  which  they  have  been  exposed  for  hours 
in  a  refrigerating  mixture  at  — 18°  C.  They  have 
even  been  subjected  by  us  to  a  temperature  of  — 175° 
C.  by  immersing  them  in  liquid  air  kept  in  an  open 
tube  for  two  hours,  and  found  to  grow  still  when  placed 
in  favorable  conditions. 

Temperatures  from  5°  to  10°  C.  over  the  optimum 
affect  bacteria  injuriously  in  several  respects.  Varieties 
are  produced  of  diminished  activity  of  growth,  the 
virulence  and  the  property  of  causing  fermentation  are 
decreased,  and  the  power  of  spore-formation  is  gradu- 
ally lost.  These  effects  may  predominate  either  in  one 
or  the  other  direction. 

If  the  maximum  temperature  is  exceeded  the  organism 
dies;  the  thermal  death- point  for  the  psychrophilic  spe- 
cies being  about  37°  C.,  for  the  mesophilic  species  about 


EFFECT  OF  TEMPERATURE  UPON  BACTERIA.     147 

45°  to  55°  C.,  and  for  the  thermophilic  species  about 
75°  C.  There  are  no  non-spore  bearing  bacteria  which 
when  moist  are  able  to  withstand  a  temperature  of 
100°  C.  even  for  a  few  minutes.  A  long  exposure  to 
temperatures  between  80°  and  100°  has  the  same  result 
as  a  shorter  one  at  the  higher  temperatures.  Accord- 
ing to  Sternberg,  ten  minutes'  exposure  to  moist  heat 
will  at  52°  C.  kill  the  cholera  spirillum,  at  54°  C.  kill 
the  streptococcus,  at  56°  C.  the  typhoid  bacillus,  at 
60°  C.  the  gonococcus,  and  at  62°  C.  the  staphylococ- 
cus,  the  latter  being  about  the  most  resistant  of  the 
pathogenic  organisms  which  have  no  spores. 

When  micro-organisms  in  a  desiccated  condition  are 
exposed  to  the  action  of  heated  dry  air  the  temperature 
required  for  their  destruction  is  much  above  that  re- 
quired when  they  are  in  a  moist  condition  or  when  they 
are  exposed  to  the  action  of  hot  water  or  steam.  A 
large  number  of  pathogenic  and  non-pathogenic  species 
are  able  to  resist  a  temperature  of  over  100°  C.  dry 
heat  for  an  hour.  A  temperature  of  120°  to  130°  C. 
maintained  for  one  and  a  half  hours  is  required  to  de- 
stroy all  bacteria,  in  the  absence  of  spores,  if  dry  heat 
is  used. 

Spores  are  far  more  resistant  to  all  injurious  influ- 
ences than  vegetative  forms.  They  retain  their  power 
of  germination  for  years  without  either  nourishment  or 
water,  and  are  much  more  indifferent  to  the  action  of 
gases  than  bacilli,  the  spores  of  the  anaerobic  species 
being  especially  resistant  to  the  action  of  oxygen. 
Spores  possess  a  great  power  of  resistance  to  both 
moist  and  dry  heat.  Dry  heat  is  comparatively  well 
borne,  many  spores  resisting  a  temperature  of  over 
130°  C.  The  spores  of  bacillus  anthracis  and  of 


148  BACTERIOLOGY. 

bacillus  subtilis  require  a  temperature  of  140°  C.  (dry 
heat)  maintained  for  three  hours  to  insure  their  de- 
struction. Moist  heat  at  a  temperature  of  100°  C., 
either  boiling  water  or  free-flowing  steam,  destroys 
the  spores  of  known  pathogenic  bacteria  within  ten 
minutes;  certain  non-pathogenic  species,  however,  re- 
sist this  temperature  for  hours.  Thus,  Globig  obtained 
a  bacillus  from  the  soil,  the  spores  of  which  required 
five  and  a  half  to  six  hours'  exposure  to  streaming 
steam  for  their  destruction.  These  spores  survived 
exposure  for  three-quarters  of  an  hour  in  steam  under 
pressure  at  from  109°  to  113°  C.  They  were  de- 
stroyed, however,  by  exposure  for  twenty-five  minutes 
in  steam  at  113°  to  116°  C.  and  in  two  minutes  at 
127°  C. 

The  resistance  of  spores  to  moist  heat  is  tested  by 
suspending  cover-glasses  upon  which  the  spores  (an- 
thrax) have  been  dried  in  little  gauze  bags  in  a  boiling 
steam  sterilizer.  The  cover-glasses  are  removed  from 
minute  to  minute  and  laid  upon  agar  plates,  which  are 
then  placed  in  the  incubator  at  37°  C.  Anthrax  spores 
are  obtained  by  carefully  removing  sporulating  streak 
cultures  on  agar  and  heating  the  emulsion  prepared 
with  a  little  water  to  70°  C.  for  five  minutes. 

In  the  practical  application  of  steam  for  disinfecting 
purposes  it  must  be  remembered  that  while  steam  under 
pressure  is  more  effective  than  streaming  steam  it  is 
scarcely  necessary  to  give  it  the  preference,  in  view  of 
the  fact  that  all  known  pathogenic  bacteria  and  their 
spores  are  quickly  destroyed  by  the  temperature  of 
boiling  water,  and  also  that  "superheated"  steam  is 
less  effective  than  moist  steam.  When  confined  steam 
in  pipes  is  "  superheated  "  it  has  about  the  same  germi- 


EFFECT  OF  TEMPERATURE  UPON  BACTERIA.     149 

cidal  power  as  hot,  dry  air  at  the  same  temperature. 
Esmarch  found  that  anthrax  spores  were  killed  in 
streaming  steam  in  four  minutes,  but  were  not  killed 
in  the  same  time  by  superheated  steam  at  a  temperature 
of  114°  C.  It  should  also  be  remembered  that  dry 
heat  has  but  little  penetrating  power.  Koch  and 
Wolffhiigel  found  that  registering  thermometers  placed 
in  the  interior  of  folded  blankets  and  packages  of  vari- 
ous kinds  did  not  show  a  temperature  capable  of  kill- 
ing bacteria  after  three  hours'  exposure  in  a  hot-air 
oven  at  133°  C.  and  over. 

Fractional  Sterilization  (Tyndalization).  Certain  nutri- 
ent media,  such  as  blood-serum  and  the  transudates  of 
the  body  cavities,  as  well  as  certain  fluid  food-stuffs, 
such  as  milk,  need  at  times  to  be  sterilized,  and  yet 
cannot  be  subjected  to  temperatures  high  enough  to 
kill  spores  without  suffering  injury.  The  property  of 
spores,  when  placed  under  suitable  conditions,  to  germi- 
nate into  the  non-spore  bearing  form,  is  here  taken 
advantage  of  by  heating  the  fluids  up  to  55°  to  70°  C. 
for  one  hour  on  each  of  six  consecutive  days.  By  this 
means  we  kill,  upon  each  exposure,  all  bacteria  in  the 
vegetative  form,  and  allow,  during  the  intervals,  for 
the  development  of  any  still  remaining  in  the  spore 
stage,  or  which  have  reproduced  spores,  to  change  again 
into  the  vegetative  form.  Experience  has  shown 
that,  with  but  few  exceptions,  an  exposure  for  six  con- 
secutive days  will  completely  sterilize  the  fluids  so 
exposed. 

Pasteurization.  It  is  sometimes  undesirable  to  expose 
food,  such  as  milk,  to  such  a  temperature  as  will  destroy 
spores,  because  of  the  deleterious  effects  of  such  high 
temperatures,  and  yet  where  a  partial  sterilization  is 


1 50  BA  CTERIOL  OGY. 

necessary.  Under  these  conditions  we  heat  the  food- 
staffs  for  thirty  minutes  to  such  a  temperature  (70°  C.) 
as  will  kill  the  bacteria  in  the  vegetative  form,  but 
allow  the  spores  to  remain  alive.  Even  this  amount 
of  sterilization  retards  greatly  the  rapidity  of  putrefac- 
tion and  fermentation. 


CHAPTER   X. 

THE  DESTRUCTION   OF   BACTERIA  BY  THE  CHEMICALS. 

MANY  chemical  substances  when  brought  in  contact 
with  bacteria  unite  with  their  cell  substance.  New 
compounds  are  thus  formed,  and  the  life  of  the  bacteria 
and  the  disinfecting  properties  of  the  substances  are  usu- 
ally destroyed.  While  in  the  vegetative  stage  bacteria 
are  much  more  easily  killed  than  when  in  the  spore 
form,  and  their  life  processes  are  inhibited  by  substances 
less  deleterious  than  those  required  to  destroy  them. 

Bacteria  both  in  the  vegetative  and  in  the  spore 
form  differ  among  themselves  considerably  in  their 
resistance  to  the  poisonous  effects  of  chemicals.  The 
reason  for  this  is  not  as  yet  clear,  but  is  apparently 
connected  with  the  structure  and  chemical  nature  of 
their  cell  substance. 

Chemicals  are  more  poisonous  at  fairly  high  than  at 
a  low  temperature,  and  act  more  quickly  upon  bacteria 
when  they  are  suspended  in  fluids  singly  than  when  in 
clumps.  The  increased  energy  of  disinfectants  at  higher 
temperatures  indicates  in  itself  a  probability  that  a  true 
chemical  reaction  takes  place.  In  estimating  the  ex- 
tent of  the  destructive  action  of  chemicals  the  follow- 
ing degrees  are  usually  distinguished: 

1.  The  growth  is  not  permanently  interfered  with, 
but  the   pathogenic  and  zymogenic  functions  of   the 
organism  are  diminished — attenuation. 

2.  The  organisms  are  not  able  to  multiply,  but  they 
are  not  destroyed  by  antiseptic  action. 


152  BACTERIOLOGY. 

3.  The  vegetative  development  of  the  organisms  is 
destroyed,  but  not  the  spores — incomplete  sterilization. 

4.  Vegetative   and  spore-formation   are   destroyed. 
This  is  complete  sterilization  or  disinfection.1 

The  methods  employed  for  the  determination  of  the 
germicidal  action  of  chemical  agents  on  bacteria  are, 
briefly,  as  follows: 

If  it  is  desired  to  determine  what  is  the  minimum  con- 
centration of  the  chemical  substance  required  to  produce 
complete  inhibition  of  growth  we  proceed  thus  :  A  10 
per  cent,  solution  of  the  disinfectant  is  prepared  and 
1  c.c.,  0.5  c.c.,  0.3  c.c.,  0.1  c.c.,  etc.,  of  this  is  added 
to  10  c.c.  of  liquefied  gelatin,  agar,  or  bouillon,  or, 
more  accurately,  10  c.c.  minus  the  amount  of  solution 
added,  in  so  many  tubes.  The  tubes  then  contain  1  per 
cent.,  0.5  per  cent.,  0.3  per  cent.,  and  0.1  per  cent,  of 
the  disinfectant.  The  fluid  med  a  in  the  tubes  are 
then  inoculated  with  a  platinum  loopful  of  the  test 
bacteria.  The  melted  agar  and  gelatin  may  be  simply 
shaken  and  allowed  to  remain  in  the  tubes,  and  watched 
as  to  whether  any  growth  takes  place,  or  the  contents 
of  the  tubes  are  poured  out  into  Petri  dishes,  where  the 
development  or  lack  of  development  of  colonies  and 
the  number  can  be  observed.  The  same  test  can  be 
made  with  material  containing  only  spores. 

If  it  is  desired  to  determine  the  degree  of  concentra- 
tion required  for  the  destruction  of  vegetative  develop- 
ment, the  organism  to  be  used  is  cultivated  in  bouillon, 
and  to  each  of  a  series  of  tubes  holding  in  watery 
solution  different  percentages  of  the  disinfectant  a 

1  Disinfection  strictly  defined  is  the  destruction  of  all  organisms  and  their 
products  which  are  capable  of  producing  disease.  Sterilization  is  the  destruc- 
tion of  all  saprophytic  as  well  as  parasitic  bacteria.  Practically,  however, 
the  two  terms  are  used  interchangeably  as  meaning  the  destruction  of  all 
living  bacteria. 


DESTE  UCTION  OF  BA  GTEEIA  B  Y  CHEMICALS.     1 53 

few  drops  of  the  culture  from  which  all  lumps  have 
been  filtered  are  added.  At  intervals  of  one,  five,  ten, 
fifteen,  and  thirty  minutes,  one  hour,  and  so  on,  a  small 
platinum  loopful  of  the  mixture  is  taken  from  each  tube 
and  inoculated  into  10  c.c.  of  lukewarm  gelatin,  from 
which  plate  cultures  are  made.  The  results  obtained  are 
signified  as  follows :  x  per  cent,  of  the  disinfectant  in 
watery  solution  and  at  x  temperature  kills  the  organism 
in  twenty  minutes,  y  per  cent,  kills  in  one  minute,  and 
so  on.  If  there  be  any  doubt  whether  the  trace  of  the 
disinfectant  carried  over  with  the  platinum  loops  may 
have  rendered  the  gelatin  unsuitable  for  growth,  thus 
falsifying  results,  control  cultures  should  be  made  with 
vigorous  bacteria  in  gelatin  to  which  a  similar  trace  of 
the  disinfectant  has  been  added. 

The  disinfectant  to  be  examined  should  always  be 
dissolved  in  an  inert  fluid,  such  as  water;  if  on  account 
of  its  being  difficultly  soluble  in  water,  it  is  necessary 
to  use  alcohol  for  its  solution,  control  experiments  may 
be  required  to  determine  the  action  of  the  alcohol  on  the 
organism.  Sometimes,  as  in  the  case  of  corrosive  sub- 
limate, the  chemical  unites  with  the  cell  substance  to 
form  an  unstable  compound,  which  inhibits  the  growth 
of  the  organism  only  while  the  union  exists.  In  some 
tests  it  is  necessary  to  break  up 'this  union  and  note 
then  whether  the  organism  is  alive  or  dead. 

In  the  above  determinations  the  absolute  strength  of 
the  disinfectant  required  is  considerably  less  when  cul- 
ture media  rich  in  albumin  are  employed  than  when  the 
opposite  is  the  case.  Thus  creoliu  (Pearsons),  when 
bouillon  is  used  as  a  culture  medium,  stops  develop- 
ment in  the  proportion  of  1  to  15,000  or  1  to  5000; 
when  ox -serum  is  used  only  in  the  proportion  of  1  to 


154 


BACTERIOLOGY. 


150  (Behring).  Cholera  spirilla  grown  in  bouillon  con- 
taining no  peptone  or  only  0.5  per  cent,  of  peptone  are 
destroyed  in  half  an  hour  by  0.1  per  cent,  of  hydro- 
chloric acid;  grown  in  2  per  cent,  peptone-bouillon 
their  vitality  is  destroyed  in  the  same  time  on  the 
addition  of  0.4  per  cent.  HC1.  In  any  case  the  organ- 
isms to  be  tested  should  all  be  treated  in  exactly  the 
same  way  and  the  results  accompanied  by  a  statement 
of  the  conditions  under  which  the  tests  were  made. 

The  following  table  gives  the  results  and  methods 
used  in  an  actual  experiment  to  test  the  effect  of  blood - 
serum  upon  the  disinfecting  action  of  bichloride  of 
mercury  and  carbolic  acid  upon  bacteria: 

TEST  FOR  THE  DIFFERENCE  OF  EFFECT  OF  BICHLORIDE  OF 
MERCURY  AND  CARBOLIC  ACID  SOLUTIONS  ON  ANTHRAX  AND 
TYPHOID  BACILLI  IN  SERUM  AND  BOUILLON. 


Time. 


A.  Serum .  .  .  .2.5  c.c. 
HgCl2  sol.  1  : 1000  2.5  c.c. 
Anthrax  thread 


B.  Bouillon  .  .  .2.5  c.c. 
HgCl2  sol.  1  : 1000  2.5  c.c. 
Anthrax  thread 


C.  Serum  .  .  .  .2.5  c.c. 
Carbolic  sol.  5  p.ct.  2.5  c.c. 
Typhoid  threads 


D.  Bouillon  .  .  .2.5  c.c. 
Carbolic  sol.  5  p.ct.  2.5  c.c. 
Typhoid  threads 


1    8    510208045hU^hr2s 


Solution  equals 

1  in  2000 

bichloride. 


Same. 


Solution  equals 
2%  per  cent, 
carbolic  acid. 


Same. 


—  Indicates  total  destruction  of  bacteria  with  no  growth  in  media. 
+  Indicates  lack  of  destruction  of  bacteria  with  growth  in  media. 


DESTR  UCTION  OF  BACTERIA  B  Y  CHEMICALS.     155 

Pieces  of  sterile  thread  (one  inch)  were  placed 
in  bouillon  cultures  of  anthrax  and  typhoid  bacilli 
for  ten  minutes,  then  removed  to  Petri  dishes,  and 
dried  in  the  incubator  for  twenty-four  hours.  These 
were  then  placed  in  serum  and  bouillon  respectively 
(2.5  c.c.).  From  each  a  control  was  taken.  Then  2.5 
c.c.  HgCl2  ( 1: 1000)  and  carbolic  solution  (5  per  cent.) 
was  added  to  either,  as  shown  in  A,  B,  C,  and  D. 
From  each  one  thread  was  taken  at  varying  periods  of 
time  and  planted  in  bouillon  tubes.  The  threads  from 
A  and  B  (HgCl2  solution)  were  washed  in  sterile  water, 
then  in  a  solution  of  ammonium  sulphide  (25  per  cent.), 
then  in  sterile  water  again,  then  in  the  bouillon.  The 
threads  from  the  carbolic  solution  were  washed  in  sterile 
water  before  planting. 

Observations:  The  serum  seems  to  have  an  inhibi- 
tory action  with  the  bichloride  solution,  allowing  a 
growth  up  to  forty-five  minutes,  while  with  bouillon 
the  action  is  much  quicker,  preventing  a  growth  after 
an  exposure  of  one  minute  or  over.  With  the  carbolic 
acid  solution  the  serum  seems  to  have  made  little  or  no 
difference  in  the  results. 

Many  substances  which  are  strong  disinfectants  be- 
come altered  under  the  conditions  in  which  they  are 
used,  so  that  they  lose  a  portion  or  all  of  their  germ- 
icidal  properties;  thus,  quicklime  and  milk  of  lime  are 
disinfecting  agents  only  so  long  as  sufficient  calcium 
hydroxide  is  present.  If  this  is  changed  by  the  carbon 
dioxide  of  the  air  into  carbonate  of  lime  it  becomes 
harmless.  Bichloride  of  mercury  and  many  other 
chemicals  form  compounds  with  many  organic  and 
inorganic  substances,  which,  though  still  germicidal, 
are  much  less  so  than  the  original  substances. 


156  BACTERIOLOGY. 

A  BRIEF  DESCRIPTION  OF  SOME  OF  THE  MORE 
COMMONLY  USED  DISINFECTANTS. 

Bichloride  of  Mercury.  This  substance,  when  present 
in  1  part  in  1,000,000  in  nutrient  gelatin  or  bouillon, 
prevents  the  development  of  parasitic  bacteria.  In 
water  1  part  in  500,000  will  kill  many  varieties  in  a 
few  minutes,  but  in  bouillon  twenty-four  hours  may 
be  needed.  With  organic  substances  its  power  is  less- 
ened, so  that  1  part  to  1000  may  be  required.  Spores 
are  killed  in  1  to  1000  watery  solution  within  one  hour. 
Corrosive  sublimate,  as  seen  in  the  figures  given  above, 
is  less  effective  as  a  germicide  in  alkaline  fluids  con- 
taining much  albuminous  substance  than  in  watery 
solution.  In  such  fluids,  beside  loss  in  other  ways, 
precipitates  of  albuminate  of  mercury  are  formed 
which  are  at  first  insoluble,  so  that  a  part  of  the  mer- 
curic salt  does  not  really  exert  any  action.  In  alkaline 
solutions,  such  as  blood,  blood- serum,  pus,  tissue-fluids, 
etc.,  the  soluble  compounds  of  mercury  are  converted 
into  oxides  or  hydroxides.  The  soluble  compounds 
can,  of  course,  remain  in  solution  only  when  there  are 
present  sufficient  quantities  of  certain  bodies  which 
render  solution  possible.  Bodies  of  this  sort  are  espe- 
cially the  alkaline  chlorides  and  iodides,  and,  above  all, 
sodium  chloride  and  ammonium  chloride.  A  very 
simple  way  of  preventing  precipitation  of  the  mer- 
cury, then,  is  to  add  a  suitable  quantity  of  common 
salt  to  the  corrosive  sublimate.  Those  compounds  of 
mercury  which,  like  the  cyanides,  are  not  precipitated 
with  alkalies,  because  they  at  once  form  double  salts, 
require  no  addition  of  salt. 

For  ordinary  use,  where  corrosive  sublimate  is  em- 


DESTR  UCTION  OF  BA  CTEEIA  B  Y  CHEMICALS.     1 57 

ployed,  solutions  of  1  to  500  and  1  to  1000  will  suffice, 
when  brought  in  contact  with  bacteria  in  that  strength, 
to  kill  the  vegetative  forms  within  fifteen  minutes, 
the  stronger  solution  to  be  used  when  much  organic 
matter  is  present. 

Biniodide  of  Mercury.  This  salt  is  very  similar  in 
its  effects  to  the  bichloride.  It  is  even  somewhat  more 
powerful. 

Nitrate  of  Silver.  Nitrate  of  silver  in  solution  has 
about  one-fourth  the  value  of  the  bichloride  of  mer- 
cury as  a  disinfectant,  but  nearly  the  same  value  in 
inhibiting  growth. 

Sulphate  of  Copper.  This  salt  has  about  5  per  cent, 
of  the  value  of  mercuric  chloride. 

Sulphate  of  Iron.     This  is  a  very  feeble  disinfectant. 

Sodium  Compounds.  A  30  per  cent,  solution  of  NaOH 
kills  anthrax  spores  in  about  ten  minutes,  and  in  4  per 
cent,  in  about  forty-five  minutes.  Sodium  carbonate 
kills  spores  with  difficulty  even  in  concentrated  solution, 
but  at  85°  C.  it  kills  spores  in  from  eight  to  ten  min- 
utes. A  5  per  cent,  solution  kills  in  a  short  time  the 
vegetative  forms  of  bacteria.  Even  ordinary  soapsuds 
have  a  slight  bactericidal  as  well  as  a  marked  cleansing 
effect.  The  bicarbonate  has  almost  no  destructive  effect 
on  bacteria. 

Calcium  Compounds.  Calcium  hydroxide  Ca(OH)2  is  a 
powerful  disinfectant;  the  carbonate,  on  the  other  hand, 
is  almost  of  no  effect.  A  1  per  cent,  watery  solution  of 
the  hydroxide  kills  bacteria  which  are  not  in  the  spore 
form  within  a  few  hours.  A  3  per  cent,  solution  kills 
typhoid  bacilli  in  one  hour.  A  20  per  cent,  solution 
added  to  equal  parts  of  feces  or  other  filth  and  mixed 
with  them  will  completely  sterilize  them  within  one  hour. 


158  BACTERIOLOGY. 

The  Effect  of  Acids.  An  amount  of  acid  which  equals 
40  c.c.  of  normal  hydrochloric  acid  per  litre  is  sufficient 
to  prevent  the  growth  of  all  varieties  of  bacteria  and 
to  kill  many.  Twice  this  amount  destroys  most  bacteria 
within  a  short  time.  The  variety  of  acid  makes  little 
difference.  Bulk  for  bulk,  the  mineral  acids  are  more 
germicidal  than  the  vegetable  acids,  but  that  is  because 
their  molecular  weight  is  so  much  less.  A  1  to  500 
solution  of  sulphuric  acid  kills  typhoid  bacilli  within 
one  hour.  Hydrochloric  acid  is  about  one-third  weaker, 
and  acetic  acid  somewhat  weaker  still.  Citric,  tartaric, 
malic,  formic,  and  salicylic  acids  are  similar  to  acetic 
acid.  Boric  acid  destroys  the  less  resistant  bacteria  in 
2  per  cent,  solution  and  inhibits  the  others. 


GASEOUS   DISINFECTANTS. 

The  germicidal  action  of  gases  is  much  more  active 
in  the  presence  of  moisture  than  in  a  dry  condition. 

Numerous  experiments  have  been  made  with  sulphur 
dioxide  gas  (SO2),  owing  to  the  fact  that  it  has  been  so 
extensively  used  for  the  disinfection  of  hospitals,  ships, 
apartments,  clothing,  etc.  This  gas  is  a  much  more 
active  germicide  in  a  moist  than  in  a  dry  condition; 
due,  no  doubt,  to  the  formation  of  the  more  active  disin- 
fecting agent — sulphurous  acid  (H2SO3).  In  a  pure  state 
anhydrous  sulphur  dioxide  does  not  destroy  spores,  and 
is  not  certain  to  destroy  bacteria  not  in  spore  form. 
Sternberg  has  shown  that  the  spores  of  the  bacillus 
anthracis  and  bacillus  subtilis  are  not  killed  by  contact 
for  some  time  with  liquid  SO2  (liquefied  by  pressure). 
Koch  found  that  various  species  of  spore-bearing  bacilli 


titiSTR UOTION  OF  BACTERIA  S  Y  CHEMICALS.    159 

exposed  for  ninety-six  hours  in  a  disinfecting  chamber 
to  the  action  of  SO2  in  the  proportion  of  from  4  to  6 
per  cent,  by  volume  were  not  destroyed.  In  the  ab- 
sence of  spores,  however,  the  anthrax  bacillus  in  a 
moist  condition,  attached  to  silk  threads,  was  found 
by  Sternberg  to  be  destroyed  in  thirty  minutes  in  an 
atmosphere  containing  1  volume  per  cent.  As  the  re- 
sult of  a  large  number  of  experiments  with  SO2  as  a 
disinfectant  it  has  been  determined  that  an  "  exposure 
for  eight  hours  to  an  atmosphere  containing  at  least 
4  volumes  per  cent,  of  this  gas  in  the  presence  of  moist- 
ure77 will  destroy  most  if  not  all  of  the  pathogenic 
bacteria  in  the  absence  of  spores. 

Peroxide  of  Hydrogen  (H2O2)  is  an  energetic  disin- 
fectant, and  in  2  per  cent,  solution  (about  40  per  cent, 
of  the  ordinary  commercial  article)  will  kill  the  spores 
of  anthrax  in  from  two  to  three  hours.  A  20  per  cent, 
solution  of  a  good  commercial  hydrogen  peroxide  solu- 
tion will  quickly  destroy  the  pyogenic  cocci  and  other 
spore-free  bacteria.  It  combines  with  organic  matter, 
becoming  inert.  It  is  prompt  in  its  action  and  not 
poisonous,  but  apt  to  deteriorate  if  not  properly 
kept. 

Chlorine  is  a  powerful  gaseous  germicide,  owing  its 
activity  to  its  affinity  for  hydrogen  and  the  consequent 
release  of  nascent  oxygen  when  it  comes  in  contact  with 
micro-organisms  in  a  moist  condition.  It  is,  there- 
fore, a  much  more  active  germicide  in  the  presence  of 
moisture  than  in  a  dry  condition.  Thus,  Fischer  and 
Proskauer  found  that  dried  anthrax  spores  exposed  for 
an  hour  in  an  atmosphere  containing  44.7  per  cent,  of 
dry  chlorine  were  not  destroyed;  but  if  the  spores  were 
previously  moistened  and  were  exposed  in  a  moist 


160  BACTERIOLOGY. 

atmosphere  for  the  same  time,  4  per  cent,  was  effective, 
and  when  the  time  was  extended  to  three  hours,  1  per 
cent,  destroyed  their  vitality.  The  anthrax  bacillus, 
in  the  absence  of  spores,  was  killed  by  exposure  in  a 
moist  atmosphere  containing  1  part  to  2500  for  twenty- 
four  hours. 

In  watery  solutions  0.2  per  cent,  kills  spores  within 
five  minutes  and  the  vegetative  forms  almost  imme- 
diately. 

Chloride  of  Lime.  The  efficacy  of  chloride  of  lime 
depends  on  the  chlorine  it  contains  in  the  form  of 
hypochlorites.  A  solution  in  water  of  0.5  to  1  per 
cent,  of  chloride  of  lime  will  kill  most  bacteria  in  one 
to  five  minutes.  A  5  per  cent,  solution  usually  de- 
stroys spores  within  one  hour. 

Bromine  and  iodine  are  of  about  the  same  value  as 
chlorine  for  gaseous  disinfectants,  in  the  moist  condition; 
but,  like  chlorine,  they  are  not  applicable  for  general 
use  in  house  disinfection,  owing  to  their  poisonous  and 
destructive  properties;  they  have  a  use  in  sewers  and 
similar  places. 

Trichloride  of  iodine  in  0.5  per  cent,  solution  de- 
stroys the  vegetative  forms  of  bacteria  in  five  minutes. 

ORGANIC   DISINFECTANTS. 

Alcohol  in  10  per  cent,  solution  inhibits  the  growth 
of  bacteria;  absolute  alcohol  kills  bacteria  in  the  vege- 
tative form  in  from  several  to  twenty-four  hours. 

Formaldehyde.  Formaldehyde,  or  formic-aldehyde, 
was  isolated  by  von  Hoffmann  in  1867,  who  obtained 
it  by  passing  the  vapors  of  methyl-alcohol  mixed  with 
air  over  finely  divided  platinum  heated  to  redness. 


DESTR  UCTION  OF  BA  CTEEIA  B  Y  CHEMICALS.     161 

The  methyl-alcohol  is  oxidized  and  produces  formal- 
dehyde as  follows: 

CH2OH  +  O  =  CH2O  —  H2O. 

Formaldehyde  is  a  gaseous  compound  having  the 
chemical  formula  CH2O  and  possessed  of  an  extremely 
irritating  odor.  At  a  temperature  of  68°  F.  the  gas 
is  polymerized — that  is  to  say,  a  second  body  is  formed, 
composed  of  a  union  of  two  molecules  of  CH2O.  This 
is  known  as  a  paraformaldehyde,  and  is  a  white,  soapy 
body,  soluble  in  boiling  water  and  alcohol;  it  exists  in 
the  solution  of  commerce — a  clear,  watery  liquid  con- 
taining from  33  to  40  per  cent,  of  the  gas  and  10  to  20 
per  cent,  of  methyl-alcohol,  its  chief  impurity.  If  the 
commercial  solution — ordinarily  known  in  the  trade  as 
"formalin" — is  evaporated  or  concentrated  above  40 
per  cent.,  paraformaldehyde  results;  and  when  this  is 
dried  in  vaccuo  over  sulphuric  acid  a  third  body — 
trioxymethylene — is  produced,  consisting  of  three  mole- 
cules of  CH2O.  This  is  a  white  powder,  almost  soluble 
in  water  or  alcohol,  and  giving  off  a  strong  odor  of 
formaldehyde.  The  solid  polymers  of  formaldehyde, 
when  heated,  are  again  reduced  to  the  gaseous  condi- 
tion; ignited,  they  finally  take  fire  and  burn  with  a 
blue  flame,  leaving  but  little  ash. 

Formaldehyde  has  an  active  affinity  for  many  organic 
substances,  and  forms  with  some  of  them  definite  chem- 
ical combinations.  It  combines  readily  with  ammonia 
to  produce  a  compound  called  ammoniacal-aldehyde, 
which  possesses  neither  the  odor  nor  the  antiseptic 
properties  of  formaldehyde.  This  action  is  made  use 
of  in  neutralizing  the  odor  of  formaldehyde  when  it  is 
desired  to  dispel  it  rapidly  after  disinfection.  Formal- 

11 


162  BACTERIOLOGY. 

dehyde  also  forms  combinations  with  certain  aniline 
colors — viz.,  fuchsin  and  safranin — the  shades  of  which 
are  thereby  changed  or  intensified.  These  are  the  only 
colors,  however,  which  are  thus  affected,  and  as  they 
are  seldom  used  in  dyeing,  owing  to  their  liability  to 
fade,  this  effect  is  of  little  practical  significance.  The 
most  delicate  fabrics  of  silk,  wool,  cotton,  fur,  leather 
etc.,  are  unaffected  in  texture  or  color  by  formaldehyde. 
Iron  and  steel  are  attacked,  after  long  exposure,  by  the 
gas,  and  more  so  by  its  solution;  but  copper,  brass, 
nickel,  zinc,  silver,  and  gilt  work  are  not  at  all  acted 
upon.  Formaldehyde  unites  with  nitrogenous  products 
of  decay — fermentation  or  decomposition — forming  true 
chemical  compounds,  which  are  odorless  and  sterile.  It 
is  thus  a  true  deodorizer  in  that  it  does  not  replace  one 
odor  by  another  more  powerful,  but  forms  new  chemical 
compounds  which  are  odorless.  Formaldehyde  has  a 
peculiar  action  upon  albumin,  which  it  transforms  into 
an  insoluble  and  indecomposable  substance.  It  ren- 
ders gelatin  insoluble  in  boiling  water  and  most  acids 
and  alkalies.  It  is  from  this  property  of  combining 
chemically  with  the  albuminoids  forming  the  protoplasm 
of  bacteria  that  formaldehyde  is  supposed  to  derive  its 
bactericidal  powers.  Formaldehyde  is  an  excellent  pre- 
servative of  organic  products.  It  has  been  proposed  to 
make  use  of  this  action  for  the  preservation  of  meat, 
milk,  and  other  food  products;  but,  according  to  Trillat 
and  other  investigators,  formaldehyde  renders  these  sub- 
stances indigestible  and  unfit  for  food.  It  has  been 
successfully  employed,  however,  as  a  preservative  of 
pathological  and  histological  specimens. 

There   are   no   exact   experiments   recorded   of   the 
physiological   action   of   formaldehyde  on   the   human 


DESTR  UCTION  OF  BA CTERIA  B  Y  CHEMICALS.     163 

subject  when  taken  internally.  Slater  and  Rideal1 
report  that  a  1  per  cent,  solution  has  been  taken  in 
considerable  quantity  without  serious  results;  and  tri- 
oxymethylene  has  been  given  in  doses  up  to  90  grains 
as  an  intestinal  antiseptic.  The  vapors  of  formalde- 
hyde are  extremely  irritating  to  the  mucous  membrane 
of  the  eyes,  nose,  and  mouth,  causing  profuse  lachry- 
mation,  coryza,  and  flow  of  saliva.  Aronson  reports 
that  in  many  of  his  experiments  rabbits  and  guinea- 
pigs  allowed  to  remain  for  twelve  and  twenty-four 
hours  in  rooms  which  were  being  disinfected  with  for- 
maldehyde gas  were  found  to  be  perfectly  well  when 
the  rooms  were  opened.  On  autopsy  the  animals 
showed  no  injurious  effects  of  the  gas.  Others  have 
noticed  that  animals,  such  as  dogs  and  cats,  which 
have  accidentally  been  confined  for  any  length  of  time 
in  rooms  undergoing  formaldehyde  disinfection  occa- 
sionally died  from  the  effects  of  the  gas.  Many 
observers,  however,  have  reported  that  insects,  such  as 
roaches,  flies,  and  bedbugs,  are  not,  as  a  rule,  affected. 
The  result  of  these  observations  would  seem  to  indicate 
that  although  formaldehyde  is  comparatively  non-toxic 
to  the  higher  forms  of  animal  life,  nevertheless  a  cer- 
tain degree  of  caution  should  be  observed  in  the  use  of 
this  agent. 

The  results  of  numerous  experiments  have  shown 
that  in  the  air,  2.5  per  cent,  by  volume  of  the  aqueous 
solution,  or  1  per  cent,  by  volume  of  the  gas,  are  suffi- 
cient to  destroy  fresh  virulent  cultures  of  the  common 
pathogenic  bacteria  in  a  few  minutes.  The  researches 
of  Pottevin  and  Trillat  have  shown  that  the  germicidal 

i  Lancet,  April  21, 1894. 


164  BACTERIOLOGY. 

power  of  the  gas  depends  not  only  upon  its  concentra- 
tion, but  also  upon  the  temperature  and  the  condition 
of  the  objects  to  be  sterilized.  As  with  other  gaseous 
disinfectants — viz.,  sulphur  dioxide  and  chlorine — it  has 
been  found  that  the  action  is  more  rapid  and  complete 
at  higher  temperatures—  i.  e.,  at  35°  to  45°  C.  (95°  to 
120°  F.) — and  when  the  test  objects  are  moist  than  at 
lower  temperatures  and  when  the  objects  are  dry.  Still 
it  has  been  repeatedly  demonstrated  by  actual  experi- 
ment in  rooms  that  it  is  possible  to  disinfect  the  surface 
of  apartments  and  articles  contained  in  them,  under  the 
conditions  of  temperature  and  moisture  ordinarily 
existing  in  rooms,  by  an  exposure  of  a  few  hours  to 
a  saturated  atmosphere  of  formaldehyde  gas. 

Stahl l  has  shown  that  bandages  and  iodoform  gauze 
can  be  kept  well  sterilized  by  placing  in  the  jars  con- 
taining them  pieces  of  <(  formolith,"  a  preparation  of 
paraformaldehyde  in  tablet  form  containing  50  per 
cent,  of  formaldehyde.  The  same  experimenter  has 
also  succeeded  in  making  carpets  and  articles  of  cloth- 
ing germ-free  by  spraying  them  with  0.5  to  2  per  cent, 
solution  of  formaldehyde  for  fifteen  to  twenty  minutes 
without  the  color  of  the  fabrics  being  in  any  way 
affected.  The  investigations  of  Trillat,  Aronson,  Pot- 
tevin,  and  others  have  shown  that  a  concentration  of 
°f  *ne  aqueous  solution  (40  per  cent.),  equal  to 
°^  Pure  formaldehyde,  was  safe  and  sufficiently 
powerful  to  retard  bacterial  growth. 

A  2  per  cent,  watery  solution  of  formalin  destroys 
the  vegetative  forms  of  bacteria  within  five  minutes. 
In  our  experiments  formalin  has  upon  the  vegetative 

1  Pharmaceutische  Zeitung,  No.  22, 1893. 


DESTR  UCTION  OF  BA CTEEIA  B  Y  CHEMICALS.     165 

forms  about  one-third  the  strength  of  pure  carbolic 
acid. 

Chloroform.  This  substance,  even  in  pure  form,  does 
not  destroy  spores,  but  it  does  bacteria  in  vegetative 
form,  even  in  1  per  cent,  solution.  Chloroform  is  used 
practically  in  sterilizing  and  keeping  sterile  blood-serum, 
which  can  be  used  later  for  culture  purposes  by  driving 
off  the  chloroform. 

lodoform.  This  substance  has  but  very  little  destruc- 
tive action  upon  bacteria;  indeed,  upon  most  varieties 
it  has  no  appreciable  effect  whatever.  When  mixed 
with  putrefying  matter,  wound  discharges,  etc.,  the 
iodoform  is  reduced  into  soluble  iodine  compounds, 
which  partly  act  destructively  upon  the  bacteria  and 
partly  unite  with  the  poisons  already  produced. 

Carbolic  Acid  (C6H5OH).  A  solution  having  1  part 
to  1000  inhibits  the  growth  of  bacteria;  1  part  to  400 
kills  the  less  resistant  bacteria,  and  1  part  to  100  kills 
the  remainder.  A  5  per  cent,  solution  kills  the  less 
resistant  spores  within  a  few  hours  and  the  more 
resistant  in  from  one  day  to  four  weeks.  A  slight 
increase  in  temperature  aids  the  destructive  action; 
thus,  even  at  37.5°  spores  are  killed  in  three  hours. 
A  3  per  cent,  solution  kills  streptococci,  staphylococci, 
anthrax  bacilli,  etc.,  within  one  minute.  Carbolic  acid 
loses  much  of  its  value  when  in  solution  in  alcohol  or 
ether.  An  addition  of  0.5  HC1  aids  its  activity. 
Carbolic  acid  is  so  permanent  and  so  comparatively 
little  influenced  by  albumin  that  it  is  rightly  widely 
used  in  practical  disinfection  even  in  places  of  more 
powerful  substances. 

Cresol  [C6H4(CH3)OH]  is  the  chief  ingredient  of  the 
so-called  "  crude  carbolic  acid."  This  is  almost  in- 


166  BACTERIOLOGY. 

soluble  in  water,  and  has,  therefore,  little  value. 
Many  methods  are  used  for  bringing  it  into  solution 
so  as  to  make  use  of  its  powerful  disinfecting  prop- 
erties. With  equal  parts  of  crude  sulphuric  acid  it  is 
a  powerful  disinfectant,  but  it  is,  of  course,  strongly 
corrosive.  An  alkaline  emulsion  of  the  cresols  and 
other  products  contained  in  "  crude'7  carbolic  acid 
with  soap  is  called  creolin.  It  is  used  in  1  to  5  per 
cent,  emulsions.  It  is  fully  as  powerful  as  pure  car- 
bolic acid.  Lysol  is  similar  to  creolin,  except  that  it 
has  more  of  the  cresols  and  less  of  the  other  products. 
It  and  creolin  are  of  about  the  same  value. 

Tricresol  is  a  refined  mixture  of  the  three  cresols 
(meta-,  para-,  and  ortho-).  It  is  soluble  in  water  to 
the  extent  of  2.5  per  cent.,  and  is  about  three  times 
the  strength  of  carbolic  acid. 

Aniline  Dyes.  Some  of  these  colors  possess  marked 
germicidal  qualities.  According  to  observers,  methyl- 
violet  (pyoktanin)  and  malachite-green  destroy  the 
typhoid  bacillus  in  bouillon  cultures  in  the  proportion 
of  1  to  200  in  two  hours'*  exposure,  and  the  pyogenic 
cocci  in  less.  In  1  to  100,000  solutions  they  are  said 
to  retard  the  development  of  bacteria. 

Oil  of  turpentine,  1  to  200,  prevents  the  growth  of 
bacteria. 

Camphor  has  very  slight  antiseptic  action. 

Creosote  in  1  to  200  kills  many  bacteria  in  ten  min- 
utes; 1  to  100  failed  to  kill  tubercle  bacilli  in  twelve 
hours. 

Essential  oils:  Cardiac  and  Meumir  found  that  the 
essences  of  cinnamon,  cloves,  thyme,  and  others  killed 
typhoid  bacilli  within  one  hour.  Sandal- wood  required 
twelve  hours. 


DESTR  UCTION  OF  BA CTERIA  B  Y  CHEMICALS.     167 

Thymol  and  eucalyptol  have  about  one-fourth  the 
strength  of  carbolic  acid  (Behring). 

Oil  of  peppermint  in  1  to  100  solution  prevents  the 
growth  of  bacteria. 

TABLE  OF  ANTISEPTIC  VALUES.1 


Alum                    .       .•      -. 

1  :  222 

Mercuric  chloride 

1    14300 

Aluminium  acetate    ,       . 
Ammonium  chloride  . 
Boric  acid             .       .      •• 

1  :  6000 
1  :9 
1  :  143 

Mercuric  iodide  . 
Potassium  bromide     . 
Potassium  iodide 

1    40000 
1    10 
1     10 

Calcium  chloride 
Calcium  hypochiorite 
Carbolic  acid       .       .       . 
Chloral  hydrate  .       . 
Cupric  sulphate  . 
Ferrous  sulphate         .  .     . 
Formaldehyde  (40  per  cent.) 
Hydrogen  peroxide     . 

1:25 
1  :1000 
1:  333 
1  :107 
1  :  2000 
1  :  200 
1  :  10000 
1  :  20000 

Potassium  permanganate  . 
Pure  formaldehyde     . 
Quinin  sulphate  . 
Silver  nitrate 
Sodium  borate 
Sodium  chloride  . 
Zinc  chloride 
Zinc  sulphate 

1    300 
1    25000 
1    800 
1    12500 
1    14 
1    6 
1    500 
1    20 

1  These  figures  are  approximately  correct,  and  represent  the  percentage  of 
disinfectant  required  to  be  added  to  a  fluid  containing  considerable  organic 
material,  in  order  to  permanently  inhibit  any  bacterial  growth. 


CHAPTEE  XI. 

PRACTICAL  DISINFECTION  AND  STERILIZATION  (HOUSE, 
PERSON,  INSTRUMENTS,  AND  FOOD) — STERILIZATION 
OF  MILK  FOR  FEEDING  INFANTS. 

DISINFECTANTS  AND  METHODS  OF  DISINFECTION 
EMPLOYED  IN  THE  HOUSE  AND  SICK-ROOM. 

Disinfection  and  Disinfectants. 

SUNLIGHT,  pure  air,  and  cleanliness  are  always  very 
important  agents  in  maintaining  health  and  in  protect- 
ing the  body  against  many  forms  of  illness.  When, 
however,  it  becomes  necessary  to  guard  against  such 
special  dangers  as  accumulated  filth  or  contagious  dis- 
eases, disinfection  is  essential.  In  order  that  disinfec- 
tion shall  afford  complete  protection  it  must  be  thorough, 
and  perfect  cleanliness  is  better,  even  in  the  presence  of 
contagious  disease,  than  filth  with  poor  disinfection. 

Since  all  forms  of  fermentation,  decomposition,  and 
putrefaction,  as  well  as  the  infectious  and  contagious 
diseases,  are  caused  by  micro-organisms,  it  is  the  object 
of  disinfection  to  kill  these.  Decomposition  and  putre- 
faction should  at  all  times  be  prevented  by  the  imme- 
diate destruction  or  removal  from  the  neighborhood  of 
the  dwelling  of  all  useless  putrescible  substances.  In 
order  that  as  few  articles  as  possible  shall  be  exposed  to 
the  germs  causing  the  contagious  diseases  and  thus  be- 
come carriers  of  infection,  it  is  important  that  all  arti- 
cles not  necessary  for  immediate  use  in  the  care  of  the 


DISINFECTION  AND  STERILIZATION.         169 

sick  person,  especially  upholstered  furniture,  carpets, 
and  curtains,  should  be  removed  from  the  room  before 
placing  in  it  the  sick  person. 

Agents  for  Cleansing  and  Disinfection. 

Too  much  emphasis  cannot  be  placed  upon  the  im- 
portance of  cleanliness,  both  as  regards  the  person  and 
the  dwelling,  in  preserving  health  and  protecting  the 
body  from  all  kinds  of  infectious  disease.  Sunlight 
and  fresh  air  should  be  freely  admitted  through  open 
windows,  and  personal  cleanliness  should  be  attained 
by  frequently  washing  the  hands  and  body. 

Cleanliness  in  dwellings,  and  in  all  places  where  men 
go,  may,  under  ordinary  circumstances,  be  well  main- 
tained by  the  use  of  the  two  following  solutions  : 

1.  Soapsuds  Solution.     For  simple  cleansing,  or  for 
cleansing  after  the  methods  of  disinfection  by  chemicals 
described  below,  one  ounce  of  common  soda  should  be 
added  to  twelve  quarts  of  hot  soap  (soft  soap)  and  water. 

2.  Strong  Soda  Solution.     This,  which  is  a  stronger 
and  more  effective  cleansing  solution  and  also  a  feeble 
disinfectant,  is  made  by  dissolving  one-half  pound  of 
common  soda  in  three  gallons  of  hot  water.     The  solu- 
tion thus  obtained  should  be  applied  by  scrubbing  with 
a  hard  brush. 

When  it  becomes  necessary  to  arrest  putrefaction  or 
to  prevent  the  spread  of  contagious  diseases  by  surely 
killing  the  living  germs  which  cause  them,  more  powerful 
agents  must  be  employed  than  those  required  for  simple 
cleanliness,  and  these  are  commonly  called  disinfectants 
The  following  are  some  of  the  most  reliable  ones: 

3.  Heat.     Complete  destruction  by  fire  is  an  abso- 
lutely safe  method  of  disposing  of  infected  articles  of 


170  BACTERIOLOGY. 

small  value,  but  continued  high  temperatures  not  as 
great  as  that  of  fire  will  destroy  all  forms  of  life;  thus, 
boiling  or  steaming  in  closed  vessels  for  one-half  hour 
will  absolutely  destroy  all  disease  germs. 

4.  Carbolic  Acid  Solution.      Dissolve  six  ounces  of 
carbolic  acid  in  one  gallon  of  hot  water.     This  makes 
approximately  a  5  per  cent,  solution  of  carbolic  acid, 
which,  for  many   purposes,   may  be   diluted  with  an 
equal  quantity  of  water.     The  commercial  "  crude  car- 
bolic acid"  should  not  be  used,  as  it  does  not  readily 
enter  into  solution.     Care  must  be  taken  that  the  pure 
acid  does  not  come  in  contact  with  the  skin. 

5.  Bichloride  Solution  (bichloride  of  mercury  or  cor- 
rosive sublimate).     Dissolve  sixty  grains  of  pulverized 
corrosive  sublimate  and  two  tablespoonfuls  of  common 
salt  in  one  gallon  of  hot  water.     This  solution  must  be 
kept  in  glass,  earthen,  or  wooden  vessels  (not  in  metal 
vessels).     For  safety  it  is  well  to  cover  the  solution. 

The  carbolic  and  bichloride  solutions  are  very  pois- 
onous when  taken  by  the  mouth,  but  are  harmless  when 
used  externally. 

6.  Milk  of  Lime.     This  mixture  is  made  by  adding 
one  quart  of  dry,  freshly  slaked  lime  to  four  or  five 
quarts  of  water.     (Lime  is  slaked  by  pouring  a  small 
quantity  of  water  on  a  lump  of  quicklime.     The  lime 
becomes  hot,  crumbles,  and  as  the  slaking  is  completed 
a  white  powder  results.     The  powder  is  used  to  make 
milk  of  lime.)     Air-slaked  lime  (the  carbonate)  has  no 
value  as  a  disinfectant. 

7.  Dry  Chloride  of  Lime.     This  must  be  fresh  and 
kept  in  closed  vessels  or  packages.     It  should  have 
the  strong,  pungent  odor  of  chlorine. 

8.  Formalin.     Add  one  part  of  formalin  to  ten  of 


DISINFECTION  AND  STERILIZATION.        171 

water.  This  equals  in  value  the  5  per  cent,  carbolic 
acid  solution. 

9.  Creolin,  Tricresol,  and  Lysol  are  of  about  the  same 
value  as  pure  carbolic  acid. 

The  proprietary  disinfectants,  which  are  so  often 
widely  advertised  and  whose  composition  is  kept  secret, 
are  relatively  expensive  and  often  unreliable  and  ineffi- 
cient. It  is  important  to  remember  that  substances 
which  destroy  or  disguise  bad  odors  are  not  necessarily 
disinfectants  and  that  there  are  very  few  disinfectants 
that  are  not  poisonous  when  taken  internally. 

[NOTE. — The  cost  of  the  carbolic  solution  is  much 
greater  than  that  of  most  of  the  other  solutions,  but 
except  for  the  disinfection  of  the  skin,  which  in  some 
persons  it  irritates,  generally  is  to  be  much  preferred 
by  those  not  thoroughly  familiar  with  disinfectants,  as 
it  does  not  deteriorate,  and  is  rather  more  uniform  in 
its  action  than  some  of  the  other  disinfectants.] 

Methods  of  Disinfection  in  Infectious  and  Contagious 
Diseases. 

The  diseases  to  be  commonly  guarded  against,  outside 
of  surgery,  by  disinfection  are  scarlet  fever,  measles, 
diphtheria,  tuberculosis,  smallpox,  typhoid  and  typhus 
fever,  yellow  fever,  and  cholera. 

1.  Hands  and  Person.  Dilute  the  carbolic  solution 
with  an  equal  amount  of  water  or  use  the  bichloride 
solution  without  dilution.  Hands  soiled  in  caring  for 
persons  suffering  from  ontagious  diseases,  or  soiled 
portions  of  the  patient's  body,  should  be  immediately 
and  thoroughly  washed  with  one  of  these  solutions  and 
then  washed  with  soap  and  water,  and  finally  immersed 


172  BACTERIOLOGY. 

again  in  the  solutions.  The  nails  should  always  be 
kept  perfectly  clean.  Before  eating  the  hands  should 
be  first  washed  in  one  of  the  above  solutions,  and  then 
thoroughly  scrubbed  with  soap  and  water  by  means  of 
a  brush. 

2.  Soiled    Clothing,    Towels,    Napkins,    Bedding,    etc., 
should  be  immediately  immersed  in  the  carbolic  solu- 
tion, in  the  sick-room,  and  soaked  for  one  or  more  hours. 
They  should  then  be  wrung  out  and  boiled  in  the  soap- 
suds solution  for  one  hour.     Articles  such  as  beds, 
woollen  clothing,  etc.,  which  cannot  be  washed,  should 
at  the  end  of  the  disease  be  referred  to  the  Health  De- 
partment, if  such  is  within  reach,  for  disinfection  or 
destruction  ;  or  if  there  is  no  public  disinfection,  these 
goods  should  be  thoroughly  exposed  to  formaldehyde 
gas,  as  noted  later. 

3.  Food  and  Drink.     Food  thoroughly  cooked  and 
drinks  that  have  been  boiled  are  free  from  disease  germs. 
Food  and  drinks,  after  cooking  or  boiling,  if  not  immedi- 
ately used,  should  be  placed  when  cool  in  clean  dishes 
or  vessels  and  covered.     In  the  presence  of  an  epidemic 
of  cholera  or  typhoid  fever,  milk  and  water  used  for 
drinking,    cooking,    washing    dishes,    etc.,    should    be 
boiled  before  using,  and  when  cholera  is  prevalent  all 
persons  should  avoid  eating  uncooked  fruit,  fresh  vege- 
tables, and  ice.     Instead  of  boiling  milk  may  be  heated 
to  80°  C.  for  one-half  hour. 

4.  Discharges  of  all  Kinds  from  the  Mouth,  Nose,  Blad- 
der, and  Bowels  of  patients   suffering  from  contagious 
diseases  should  be  received  into  glass  or  earthen  vessels 
containing  the  carbolic  or  bichloride  of  mercury  solu- 
tion, or  milk  of  lime,  or  they  should  be  removed  on 
pieces  of  cloth,  which  are  immediately  immersed  in  one 


DISINFECTION  AND  STERILI2A  TION.        1 73 

of  these  solutions.  Special  care  should  be  observed  to 
disinfect  at  once  the  vomited  matter  and  the  intestinal 
discharges  from  cholera  patients.  In  typhoid  fever 
the  urine  and  the  intestinal  discharges,  and  in  diph- 
theria, measles,  and  scarlet  fever  the  discharges  from 
the  throat  and  nose,  all  carry  infection,  and  should  be 
treated  in  the  same  manner.  The  volume  of  the  solu- 
tion used  to  disinfect  discharges  should  be  at  least  twice 
as  great  as  that  of  the  discharge.  After  standing  for 
an  hour  or  more  the  disinfecting  solution  with  the  dis- 
charges may  be  thrown  into  the  water-closet.  Cloths, 
towels,  napkins,  bedding,  or  clothing  soiled  by  the  dis- 
charges must  be  at  once  placed  in  the  carbolic  solution 
and  the  hands  of  the  attendants  disinfected,  as  described 
above.  In  convalescence  from  measles  and  scarlet  fever 
the  scales  from  the  skin  are  also  carriers  of  infection. 
To  prevent  the  dissemination  of  disease  by  means  of 
these  scales  the  skin  should  be  carefully  washed  daily  in 
warm  soap  and  water.  After  use  the  soapsuds  should 
be  disinfected  and  thrown  into  the  water-closet. 

Masses  of  feces  are  extremely  difficult  to  disinfect 
except  on  the  surface,  for  it  takes  disinfectants  such  as 
the  carbolic  acid  solution  some  twelve  hours  to  pene- 
trate to  their  interior.  If  fecal  masses  are  to  be  thrown 
into  places  where  the  disinfectant  solution  covering 
them  will  be  washed  off,  it  will  be  necessary  to  be  cer- 
tain that  the  disinfectant  has  previously  penetrated  to 
all  portions  and  destroyed  the  disease  germs.  This  can 
be  brought  about  by  stirring  them  up  with  the  disinfec- 
tant and  allowing  the  mixture  to  stand  for  one  hour,  or 
by  washing  them  into  a  pot  holding  soda  solution  which 
is  already  at  the  boiling  temperature,  or  later  will  be 
brought  to  one. 


174  BACTERIOLOGY. 

5.  The  Sputum  from  Consumptive  Patients.      The  im- 
portance of  the  proper  disinfection  of  the  sputum  from 
consumptive    patients   is   still    underestimated.      Con- 
sumption is  an   infectious  disease,  and  is  always  the 
result  of  transmission  from  the  sick  to  the  healthy  or 
from  animals  to  man.     The  sputum  contains  the  germs 
which  cause  the  disease,  and  in  a  large  proportion  of 
rases  is  the  source  of  infection.    After  being  discharged, 
unless  properly  disposed  of,  it  may  become  dry  and  pul- 
verized and  float  in  the  air  as  dust.     This  dust  con- 
tains the  germs,  and  is  a  common  cause  of  the  disease, 
through  inhalation.     In  all  cases,  therefore,  the  sputum 
should  be  disinfected  when  discharged.     It  should  be 
received  in  covered  cups  containing  the  carbolic  or  milk 
of  lime  solution.     Handkerchiefs  soiled  by  it  should  be 
soaked  in  the  carbolic  solution  and  then  boiled.     Dust 
from  the  walls,  mouldings,  pictures,  etc.,  in  rooms  that 
have  been  occupied  by  consumptive  patients,  where  the 
rules  of  cleanliness  have  not  been  carried  out,  contain 
the  germs  and  will  produce  tuberculosis  in  animals  when 
used  for  their  inoculation ;  therefore,  rooms  should  be 
thoroughly  disinfected  before  they  are  again  occupied. 
If  the  sputum  of  all  consumptive  patients  were  de- 
stroyed at  once  when  discharged  a  large  proportion  of 
the  cases  of  the  disease  would  be  prevented. 

6.  Closets,  Kitchen  and  Hallway  Sinks,  etc.    The  closet 
should  never  be  used  for  infected  discharges  until  they 
have  been  thoroughly  disinfected,  if  it  can  be  avoided; 
if  done,  one  pint  of  carbolic  solution  should  be  poured 
into  the  pan  (after  it  is  emptied)  and  allowed  to  remain 
there.     Sinks  should  be  flushed  at  least  once  daily. 

7.  Dishes,  Knives,  Forks,  Spoons,  etc.,  used  by  a  patient 
should,  as  a  rule,  be  kept  for  his  exclusive  use  and  not 


DISINFECTION  AND  STERILIZATION.        175 

removed  from  the  room.  They  should  be  washed  first 
in  the  carbolic  solution,  then  in  boiling  hot  soapsuds, 
and  finally  rinsed  in  hot  water.  These  washing  fluids 
should  afterward  be  thrown  into  the  water-closet.  The 
remains  of  the  patient's  meals  may  be  burned  or  thrown 
into  a  vessel  containing  the  carbolic  solution  or  milk  of 
lime,  and  allowed  to  stand  for  one  hour  before  being 
thrown  away. 

8.  Rooms  and  Their  Contents.     Rooms  which  have 
been   occupied   by  persons  suffering   from  contagious 
disease  should  not  be  again  occupied  until  they  have 
been  thoroughly  disinfected.     For  this  purpose  either 
careful  fumigation  with  formaldehyde  gas  or  sulphur 
should  be  employed,  or  this  combined  with  the  following 
procedure  :  Carpets,  curtains,  and  upholstered  furniture 
which  have  been  soiled  by  discharges,  or  which  have 
been  exposed  to  infection  in  the  room  during  the  illness, 
will  be  removed  for  disinfection  to  chambers  where  they 
can    be  exposed  to  formaldehyde   gas   and   moderate 
warmth  for  twelve  to  twenty-four  hours,  or  to  steam. 
Woodwork,  floors,  and  plain  furniture  will   be  thor- 
oughly washed  with  the  soapsuds  and  bichloride  solu- 
tions. 

9.  Rags,  Cloths,  and  Articles  of  Small  Value,  which 
have  been  soiled   by  discharges  or  infected  in  other 
ways,  should  be  boiled  or  burned. 

10.  In  Case  of  Death,  the  body  should  be  completely 
wrapped  in  several  thicknesses  of  cloth  wrung  out  of 
the  carbolic  or  bichloride  solution,  and  when  possible 
placed  in  a  hermetically  sealed  coffin. 

It  is  important  to  remember  that  an  abundance  of 
fresh  a//-,  sunlight,  and  absolute  cleanliness  not  only 
helps  protect  the  attendants  from  infection  and  aid  in 


176  BACTERIOLOGY. 

the  recovery  of  the  sick,  but  directly  destroys  the  bac- 
teria which  cause  disease. 

Methods  of  Cleanliness  and  Disinfection  to  Prevent  the 
Occurrence  of  Illness. 

1.  Water-closet  Bowls  and  all  Receptacles  for  Human 
Excrement  should  be  kept  perfectly  clean  by  frequent 
flushing  with  a  large  quantity  of  water,  and  as  often  as 
necessary  disinfected  with  the  carbolic  or  bichloride  solu- 
tions.    The  woodwork  around  and  beneath  them  should 
be  frequently  scrubbed  with  the  hot  soapsuds  solution. 

2.  Sinks  and  the  Woodwork  Around  and  the   Floor 
Beneath   them    should   be   frequently  and   thoroughly 
scrubbed  with  the  hot  soapsuds  solution. 

3.  School  Sinks.     School  sinks  should  be  thoroughly 
flushed  with  a  large  quantity  of  water  at  least  twice 
daily,  and  should  be  carefully  cleaned  twice  a  week  or 
oftener  by  scrubbing.     Several  quarts  of  the  carbolic 
solution  should  be  frequently  thrown  in  the  sink  after 
it  has  been  flushed. 

4.  Cesspools   and  Privy  Vaults.     An    abundance    of 
milk  of  lime  or  chloride  of  lime  should  be  thrown  into 
these  daily,  and   their  contents   should  be  frequently 
removed. 

5.  Cellars  and  Rooms  in  Cellars  are  to  be  frequently 
whitewashed,  and,   if   necessary,  the  floors  sprinkled 
with  dry  chloride  of  lime.     Areas  and  paved  yards 
should  be  cleaned,  scrubbed,  and,  if  necessary,  washed 
with  the  bichloride  solution.     Street  gutters  and  drains 
should  be  cleaned,  and,  when  necessary,  sprinkled  with 
chloride  of  lime  or  washed  with  milk  of  lime. 

6.  Air-shafts.     Air-shafts    should    be   first   cleaned 
thoroughly  and  then  whitewashed.     To  prevent  ten- 


DISINFECTION  AND  STERILIZATION.        177 

ants  throwing  garbage  down  air-shafts  it  is  sometimes 
advisable  to  put  wire  netting  outside  of  windows  open- 
ing on  shafts.  Concrete  or  asphalt  bottoms  of  shafts 
should  be  cleaned  and  washed  with  the  bichloride  solu- 
tion or  sprinkled  with  chloride  of  lime. 

7.  Hydrant  Sinks,  Garbage  Receptacles,  and  Garbage 
and    Oyster-shell    Shutes    and    Receptacles    should    be 
cleaned  daily  and  sprinkled  with  dry  chloride  of  lime. 

8.  Refrigerators  and  the  Surfaces  Around  and  Beneath 
Them,  Dumb-waiters,  etc.,  may  be  cleaned  by  scrubbing 
them  with  the  hot  soapsuds  solution. 

9.  Traps.     All  traps  should  be  flushed  daily  with  an 
abundance  of  water.     If  at  any  time  they  become  foul 
they  may  be  cleaned  by  pouring  considerable  quantities 
of  the  hot  strong  soda  solution  into  them,  followed  by 
the  carbolic  solution. 

10.  Urinals  and  the  Floors  Around  and  Underneath 
them  should  be  cleaned  twice  daily  with  the  hot  soap- 
suds solution,  and  in  addition  to  this,  if  offensive,  they 
may  be  disinfected  with  the  carbolic  solution. 

11.  Stable  Floors  and  Manure  Vaults.     Stable  floors 
should  be  kept  clean  and  occasionally  washed  with  the 
hot  soapsuds  or  the  hot  strong  soda  solution.     Pow- 
dered fresh  chloride  of  lime  or  formalin  may  be  used 
in  manure  vaults. 

12.  Vacant  Rooms  should  be  frequently  aired. 

13.  The  Woodwork  in  School-houses  should  be  scrubbed 
weekly  with  hot  soapsuds.     This  refers  to  floors,  doors, 
door-handles,  and  all  woodwork  touched  by  the  scholars7 
hands. 

14.  Spittoons  in  all  Public  Places  should  be  emptied 
daily  and  washed  with  the  hot  soapsuds  solution,  after 
which  a  small  quantity  of  the  carbolic  solution  or  milk 

12 


178  BACTERIOLOGY. 

of  lime  should  be  put  in  the  vessel  to  receive  the  expec- 
toration. 

15.  Elevated  and  Surface  Cars,  Ferry-boats,  and  Public 
Conveyances.  The  floors,  door-handles,  railings,  and 
all  parts  touched  by  the  hands  of  passengers  should  be 
washed  frequently  with  the  hot  soapsuds  solution.  Slat- 
mats  from  cars,  etc.,  should  be  cleaned  by  scrubbing 
with  a  stiff  brush  in  the  hot  soapsuds  solution. 

Use  of  Bromine  Solution  as  a  Deodorant.  Slaughter- 
houses, butchers'  ice-boxes  and  wagons,  trenches,  excava- 
tions, stable  floors,  manure-vaults,  dead  animals,  offal, 
offal  docks,  etc.,  may  be  deodorized  by  a  weak  solution 
of  bromine,  which  is  a  valuable  agent  for  this  purpose. 
The  bromine  solution,  however,  is  only  temporary  in 
its  action,  and  must  be  used  repeatedly.  It  should  be 
applied  by  sprinkling.  Although  somewhat  corrosive 
in  its  action  on  metals,  it  is  otherwise  harmless. 

The  solution  of  bromine  must  be  prepared  with  great 
care,  as  the  pure  bromine  from  which  it  is  made  is  dan- 
gerous. It  is  very  caustic  when  brought  in  contact  with 
the  skin;  it  is  volatile  and  its  fumes  are  very  irritating 
when  inhaled.  To  prepare  the  solution  an  ounce  bottle 
of  liquid  bromine  is  dropped  into  three  gallons  of  water, 
and  broken  under  the  water  and  thoroughly  stirred. 

The  Practical  Employment  of  Formaldehyde  and  Sulphur 
Dioxide  Gases  in  the  surface  disinfection  of  rooms  and  the 
disinfection  of  goods  which  would  be  injured  by  heat. 
Formaldehyde  gas  has  so  recently  come  into  use,  and  is 
for  many  purposes  so  valuable,  that  the  description  of 
methods  employed  to  generate  and  use  it  will  be  given 
in  detail. 

If  we  consider  now  the  practical  application  of 
formaldehyde  gas  for  purposes  of  disinfection  we  find 


DISINFECTION  AND  STERILIZATION.        179 

that  its  destructive  action  on  micro-organisms  depends 
upon  a  number  of  factors,  the  chief  of  which  are  its 
concentration  in  the  surrounding  atmosphere,  the  length 
of  the  contact,  the  existing  temperature,  the  accompany, 
ing  moisture,  and  the  nature  of  the  organism. 

The  necessary  concentration  of  the  gas  in  the  sur- 
rounding atmosphere  to  kill  the  micro-organisms  varies 
with  each  species,  for  some  resist  chemical  agents  much 
more  than  others,  and  also  with  the  freedom  of  access 
of  the  gas  to  the  bacteria,  for  if  they  are  under  cover 
or  within  fabrics  a  greater  amount  of  gas  must  be  gen- 
erated than  if  they  are  freely  exposed. 

For  purely  surface  disinfection,  when  the  less  resistant 
bacteria  or  other  micro-organisms  are  to  be  destroyed, 
there  will  be  required,  according  to  the  method  used, 
6  to  10  ounces  of  formalin  of  full  strength,  or  its  equiv- 
alent, to  1000  cubic  feet. 

For  the  destruction  of  the  more  resistant,  but  non- 
spore  bearing  forms,  such  as  typhoid  fever  or  tubercle 
bacilli,  at  least  twelve  ounces  of  formalin  should  be  used. 
The  gas  penetrates  through  fabrics  with  difficulty,  and 
to  pass  through  heavy  goods  the  concentration  of  the 
gas  must  be  doubled  and  heat  added. 

The  Value  of  Moisture.  At  first  it  was  thought  that 
formaldehyde  gas  acted  more  effectually  in  a  dry  atmos- 
phere, but  further  investigation  has  proved  that  although 
it  does  destroy  bacteria  with  the  amount  of  moisture 
usually  present  in  the  air,  and  contained  in  their  own 
substance,  yet  it  acts  much  more  powerfully  and  cer- 
tainly when  additional  moisture  is  present,  and  best 
when  present  up  to  the  point  of  saturation.  The 
actual  spraying  of  walls  and  goods  to  be  disinfected 
with  water  is  even  more  efficacious. 


180  BACTERIOLOGY. 

A  fairly  high /temperature — but  one  still  below  that 
which  would  injure  delicate  fabrics — increases  not  only 
the  activity  of  formaldehyde  gas  but  also  its  penetra- 
tive power,  and  for  heavy  goods  it  is  essential.  The 
production  of  a  partial  vacuum  in  the  chambers  be- 
fore the  introduction  of  the  formaldehyde  gas  still 
further  assists  its  penetration. 

The  length  of  exposure  necessary  for  complete  dis- 
infection depends  upon  the  nature  of  the  disease  for 
which  it  is  carried  out — the  penetration  required,  the 
concentration  of  the  gas  used,  the  amount  of  moisture 
in  the  air,  the  temperature  of  the  air,  and  the  size  and 
shape  of  the  room.  For  surface  disinfection  in  rooms, 
when  as  much  as  12  ounces  of  formalin  are  used  for 
each  1000  cubic  feet,  five  hours'  exposure  is  amply 
sufficient,  most  bacteria  being  killed  within  the  first 
few  minutes.  For  the  destruction  of  micro-organisms 
protected  by  even  a  layer  of  thin  covering,  double  the 
formalin  and  double  the  time  of  exposure  should  be 
allowed,  and  even  then  the  killing  of  many  species  of 
non-spore  bearing  bacteria  cannot  be  counted  upon 
in  ordinary  rooms.  When  absolutely  complete  disinfec- 
tion is  demanded,  where  penetration  of  gas  is  required, 
the  goods  must  be  placed  in  chambers  where  moderate 
heat  can  be  added  and  all  leakage  of  gas  prevented. 

Various  forms  of  apparatus  can  be  properly  employed 
to  liberate  formaldehyde  gas  for  purposes  of  disinfec- 
tion, as  each  of  these  is  lauded  by  its  maker  as  the 
best;  it  may  be  of  interest  to  give  the  results  obtained 
by  us  from  those  in  most  common  use.  There  are  two 
essentials  to  any  good  method — namely,  that  the  for- 
maldehyde gas  is  given  off  quickly;  and  that  there  is 
no  great  loss  by  deterioration  of  the  formalin. 


DISINFECTION  AND  STERILIZATION.        181 

From  Wood  Alcohol.  A  number  of  lamps  have  been 
devised,  all  very  much  on  the  same  principle,  though 
varying  somewhat  in  mechanical  construction,  which 
bring  about  the  incomplete  oxidation  of  methyl-alcohol 
by  passing  the  vapors  mixed  with  air  over  the  incan- 
descent metal.  Although  disinfection  can  be  carried 
out  by  the  best  of  these  lamps,  in  our  experience  none 
of  them  up  to  the  present  time  are  satisfactory  or  eco- 
nomical. They  may  be  very  useful  as  deodorizers  in 
the  sick-room  or  other  places. 

In  spite  of  present  failures,  it  is,  however,  probable 
that  in  the  future  this  method  may  become  practicable. 

From  Formochloral  by  the  Trillat  System.  This  sys- 
tem consists  in  heating,  under  three  atmospheres  of 
pressure,  a  solution  of  formaldehyde  gas  in  water 
mixed  with  30  per  cent,  of  calcium  chloride,  known 
as  "  forrnochloral,"  to  a  temperature  of  135°  C. 
(255°  F.).  It  is  claimed  for  this  method  of  pro- 
ducing the  gas  from  formochloral  that  the  polymeri- 
zation of  the  formaldehyde  is  prevented,  which  would 
otherwise  take  place  if  a  solution  of  formaldehyde  were 
evaporated  under  ordinary  conditions,  and  that  thereby 
the  whole  of  the  formaldehyde  is  obtained  in  the  gas- 
eous state.  The  addition  of  any  neutral  salt  aids  the 
process,  it  is  said,  but  calcium  chloride  is  the  best. 
The  results  with  this  apparatus  have  been  satisfactory, 
but  not  more  so  than  by  other  methods.  The  appa- 
ratus is  expensive  and  heavy. 

From  Formalin  by  the  New  York  Sanitary  Construction 
Company's  System.  This  system  consists  in  heating 
the  ordinary  commercial  formalin  to  a  temperature  of 
about  1000°  F.  in  an  incandescent  copper  coil  or 
chamber,  and  allowing  the  vapors  to  pass  off  freely. 


182  BACTERIOLOGY. 

It  is  claimed  for  this  method  that  the  degree  of  heat 
necessary  to  break  up  the  polymerized  products  formed 
is  supplied,  and  thus  a  loss  of  formaldehyde  is  pre- 
vented. A  further  action  of  the  intense  heat  in  the 
copper  tube  on  the  solution  is  to  partially  convert  the 
methyl-alcohol  contained  in  commercial  formalin  into 
formaldehyde  gas  by  partial  oxidation,  thereby  pre- 
venting the  formation  of  methylal  and  increasing  the 
amount  of  formaldehyde. 

The  apparatus  consists  of  a  closed  receiver  of  copper 
holding  about  a  gallon,  a  coil  of  copper  pipe  attached 
at  one  end  to  the  bottom  of  the  receiver,  and,  like  the 
preceding  apparatus  and  that  made  by  Lentz,  at  the 
other,  by  means  of  a  suitable  connection  (rubber  tube 
with  gutta-percha  or  metallic  mouth-piece),  with  the 
room  or  apartment  to  be  disinfected,  and  a  heating 
lamp  (Swedish  lamp  or  Bunsen  burner).  In  opera- 
tion the  desired  quantity  of  formalin  is  placed  in  the 
receiver  and  the  receiver  is  closed.  The  lamp  is 
lighted  and  the  coil  brought  to  a  red  heat.  The  valve 
is  then  opened  and  the  solution  contained  in  the  receiver 
is  allowed  to  pass  down  and  into  the  coil  in  a  fine 
stream.  Upon  coming  in  contact  with  the  heated 
metal  the  formaldehyde  solution  is  instantly  decom- 
posed, and  the  liberated  gas  is  further  purified  as  it 
progresses  through  the  incandescent  coil.  The  re- 
sults with  this  apparatus  have  been  as  good  as  those 
obtained  by  the  Trillat  or  Lentz  systems.  The 
apparatus  is  liable  to  get  out  of  order,  in  that  the 
valve  is  apt  to  become  clogged  and  so  stop  the  flow 
of  formalin  until  freed  by  a  wire  supplied  for  the 
purpose. 

A  great  improvement  in  this  apparatus  has  recently 


DISINFECTION  AND  STERILIZATION.        183 

been  made  by  the  originator  of  the  previous  apparatus, 
Mr.  Taylor,  partly  through  our  suggestions. 

In  the  new  form  (Fig.  15)  the  formalin  is  first  boiled 
in  the  large  chamber  and  passes  as  vapor  through  the 
tube  connecting  B  and  C.  In  C  it  is  superheated  and 
passes  out  the  tube  D  into  the  room.  In  this  apparatus 

FIG.  15. 


Formaldehyde  apparatus. 

there  is  nothing  to  get  out  of  order,  and  it  operates 
quickly.  Up  to  the  present  time  this  is  the  most  prac- 
tical apparatus  we  have  met  with,  when  the  initial  cost, 
about  $25.00,  is  not  an  objection.  It  is  handled  by 
H.  K.  Mulford  Company,  Philadelphia.  In  all  forms 
of  apparatus  where  formalin  is  used  the  large  receiving 


184  BACTERIOLOGY. 

chamber  should  be  washed  out  from  time  to  time  with 
hot  water,  to  remove  any  deposit  there  may  be. 

From  Trioxymethylene  by  Schering's  System.  This  sys- 
tem consists  in  heating  the  solid  polymer  of  formalde- 
hyde (trioxymethylene)  in  a  lamp  specially  constructed 
for  the  purpose  by  the  Chemische  Fabrik  auf  Actien, 
in  Berlin.  The  trioxymethylene  is  used  in  the  form  of 
compressed  tablets  or  pastilles,  as  being  more  convenient 
for  use.  Each  pastille  contains  the  equivalent  of  100 
per  cent,  of  formaldehyde  gas,  according  to  the  manu- 
facturers, and  weighs  1  gramme. 

The  mode  of  using  the  apparatus  is  very  simple: 
The  disinfector  is  placed  upon  a  sheet  of  iron  on  the 
floor  of  the  room  to  be  disinfected.  From  100  to  250 
pastilles  can  be  evaporated  at  a  time  in  the  apparatus. 
For  the  production  of  greater  quantities  of  formalde- 
hyde vapor  several  of  these  outfits  may  be  used 
together.  The  lamp  is  filled  with  ordinary  or  wood 
alcohol,  about  twice  as  many  cubic  centimetres  of  the 
alcohol  being  employed  as  there  are  pastilles  to  be  evap- 
orated. The  wicks  should  project  but  little  above  the 
necks  of  the  burners,  or  the  apparatus  may  get  too  hot 
and  ignite  the  pastilles.  The  vessel  is  charged  with 
formalin  pastilles  and  the  disinfector  placed  over  the 
lighted  spirit  lamp.  The  lamp  is  then  allowed  to  burn 
out  in  the  closed  room.  One  hundred  pastilles  are  con- 
sidered to  be  sufficient  for  the  disinfection  of  1000  cubic 
feet  of  space.  Lately,  a  small  steam  boiler  has  been 
added  to  the  apparatus,  for  the  purpose  of  furnishing 
sufficient  moisture  with  the  gas.  The  results  obtained 
by  us  in  superficial  disinfection,  when  from  150  to  200 
pastilles  have  been  used  to  each  1000  cubic  feet,  have 
been  good.  The  great  advantage  of  the  method  is  in  the 


DISINFECTION  AND  STERILIZATION.        185 

small  cost  of  the  apparatus,  $3.00,  and  the  avoidance 
of  the  danger  of  deterioration,  which  is  present  to  some 
extent  in  formalin.  Smaller  lamps  are  very  useful  for 
the  deodorization  of  rooms. 

From  Formalin  to  which  Glycerin  has  been  Added. 
A  very  convenient  apparatus  of  somewhat  greater  cost 
than  that  of  Sobering' s  is  prepared  by  Charles  Lentz  & 
Sons,  of  Philadelphia.  To  the  formalin  is  added  10 
per  cent,  of  glycerin,  and  the  mixture  is  simply  boiled 
iu  a  suitable  copper  vessel,  the  steam  and  formaldehyde 
gas  passing  off  by  a  tube.  This  is  a  very  serviceable 
apparatus.  When  it  is  attempted  to  vaporize  the  for- 
malin too  rapidly  part  of  it  passes  over  in  fluid  form, 
and  is  thus  wasted. 

With  a  slightly  greater  amount  of  formalin  than  that 
used  in  the  high  temperature  autoclave  and  heated  tube 
or  chamber  methods  the  results  seem  to  be  equally  as 
good.  The  apparatus  is  very  easy  to  use,  and  not  liable 
to  get  out  of  order. 

Similar  forms  of  apparatus  are  also  employed,  when 
instead  of  glycerin  the  formalin  is  mixed  with  an 
equal  quantity  of  water.  The  water  is  for  the  purpose 
of  giving  additional  moisture  to  the  air,  and,  at  the 
same  time,  like  the  glycerin,  to  prevent  the  change  of 
formaldehyde  into  inert  substances.  A  still  simpler 
method  is  to  hang  sheets  in  a  room  and  throw  on  them 
six  to  twelve  ounces  of  formalin  for  each  1000  cubic 
feet,  and  leave  for  six  hours.  If  the  room  is  tightly 
sealed  very  fair  superficial  disinfection  will  take 
place. 

As  a  result  of  the  investigations  undertaken  in  the 
department  of  health  laboratories  on  the  use  of  form- 
aldehyde as  a  disinfectant,  and  a  consideration  of  the 


186  BACTERIOLOGY. 

work  of  others,  the  conclusions  reached  by  us  may  be 
summarized  as  follows  : 

1.   Disinfection  of  Infected  Dwellings. 

Exposed  surfaces  of  walls,  *  carpets,  hangings,  etc., 
in  rooms  may  be  superficially  disinfected  by  means 
of  formaldehyde  gas.  All  apertures  in  the  rooms  should 
be  tightly  closed  and  from  6  to  12  ounces  of  formalin 
or  its  equivalent  used  to  generate  the  gas  for  each  1000 
cubic  feet.  The  time  of  exposure  should  be  not  less 
than  four  hours,  and  a  suitable  apparatus  should  be 
employed.  The  temperature  of  the  apartment  should 
be  as  high  as  possible,  and  certainly  not  below  52°  F. 
When  generated  very  rapidly  the  formaldehyde  gives 
much  better  results  than  when  giveu  off  slowly. 

Under  these  conditions  spore-free  bacteria  and  the 
contagion  of  the  exanthemata  are  surely  destroyed  when 
freely  exposed  to  the  action  of  the  gas.  Spore-bearing 
bacteria  are  not  thus  generally  destroyed;  but  these 
latter  are  of  such  rare  occurrence  in  disease,  that  in 
house  disinfection  they  may  usually  be  disregarded, 
and,  if  present,  special  measures  can  be  taken. 

The  penetrative  power  of  formaldehyde  gas  in  the 
ordinary  room,  at  the  usual  temperature,  even  when 
used  in  double  the  strength  necessary  for  surface  disin- 
fection, is  extremely  limited,  not  passing,  as  a  rule, 
through  more  than  one  layer  of  cloth  of  medium  thick- 
ness. Articles,  therefore,  such  as  bedding,  carpets, 
upholstery,  clothing,  and  the  like,  should,  when  pos- 
sible, be  subjected  to  steam,  hot  air,  or  formaldehyde 
disinfection  in  special  chambers  constructed  for  the 
purpose.  If  not,  they  must  be  thoroughly  exposed  on 
all  sides. 


DISINFECTION  AND  STERILIZATION.        187 

2.  Disinfection  of  Bedding,  Carpets,  Upholstery,  Etc. 

Bedding,  carpets,  clothing,  etc.,  which  would  be  in- 
jured by  steam,  may  be  disinfected  by  means  of  formal- 
dehyde gas  in  an  ordinary  steam  disinfecting  chamber, 
the  latter  to  be  provided  with  a  heating  and  if  pos- 
sible a  vacuum  apparatus  and  special  apparatus  for 
generating  the  gas.  Where  penetration  through  heavy 
articles  is  required  the  gas  should  be  used  in  the  pro- 
portion of  not  less  than  the  amount  derived  from  30 
ounces  of  formalin  for  each  1000  cubic  feet,  the  time 
of  exposure  to  be  not  less  than  eight  hours  and  the 
temperature  of  the  chamber  not  below  110°  F. 

In  order  to  insure  complete  sterilization  of  the  articles 
they  should  be  so  placed  as  to  allow  of  a  free  circulation 
of  the  gas  around  them — that  is,  in  the  case  of  bedding, 
clothing,  etc.,  these  should  either  be  spread  out  on  per- 
forated wire  shelves  or  loosely  suspended  in  the  cham- 
ber. The  aid  of  a  partial  vacuum  facilitates  the  opera- 
tion. Upholstered  furniture  and  articles  requiring  much 
space  should  be  placed  in  a  large  chamber,  or,  better,  in 
a  room  which  can.be  heated  to  the  required  temperature. 

The  most  delicate  fabrics,  furs,  leather,  and  other 
articles,  which  are  injured  by  steam,  hot  air  at  230°  F., 
or  other  disinfectants,  are  unaffected  by  formaldehyde. 

3.  Disinfection  of  Books. 

Books  may  be  satisfactorily  disinfected  by  means  of 
formaldehyde  gas  in  a  special  room,  or  in  the  ordinary 
steam  chamber,  as  above  described,  and  under  the  same 
condition  of  volume  of  gas,  temperature,  and  time  of 
exposure.  The  books  should  be  arranged  to  stand  as 
widely  open  as  possible  upon  perforated  wire  shelves, 


188  BACTERIOLOGY. 

set  about  one  or  one  and  a  half  feet  apart  in  the  chamber. 
A  chamber  having  a  capacity  of  200  to  250  cubic  feet 
would  thus  afford  accommodation  for  about  one  hundred 
books  at  a  time. 

Books,  with  the  exception  of  their  surfaces,  cannot 
be  satisfactorily  disinfected  by  formaldehyde  gas  in  the 
book-cases  of  houses  and  libraries,  or  anywhere  except 
in  special  chambers  constructed  for  the  purpose,  because 
the  conditions  required  for  their  thorough  disinfection 
cannot  otherwise  be  complied  with. 

The  bindings,  illustrations,  and  print  of  books  are  in 
no  way  affected  by  the  action  of  formaldehyde  gas. 

4.  Disinfection  of  Carriages,  Etc. 

Carriages,  ambulances,  cars,  etc.,  can  be  easily  disin- 
fected by  having  built  a  small,  tight  building,  in  which 
they  are  enclosed  and  surrounded  with  formaldehyde 
gas.  Such  a  building  is  used  for  disinfecting  ambu- 
lances in  New  York  City.  With  the  apparatus  there 
employed  a  large  amount  of  formalin  is  rapidly  vapor- 
ized, and  superficial  disinfection  is  completed  in  thirty 
minutes. 

5.  Advantages  of  Formaldehyde  Gas  over  Sulphur  Dioxide 
for  the  Disinfection  of  Dwellings. 

Formaldehyde  gas  is  superior  to  sulphur  dioxide  as 
a  disinfectant  for  dwellings,  first,  because  it  is  more 
efficient  in  its  action;  second,  because  it  is  less  inju- 
rious in  its  effects  on  household  goods;  third,  because 
when  necessary  it  can  easily  be  supplied  from  a  gen- 
erator placed  outside  of  the  room  and  watched  by  an 
attendant,  thus  avoiding  in  some  cases  danger  of  fire. 

Apart  from  the  cost  of  the  apparatus  and  the  greater 


DISINFECTION  AND  STERILIZATION.        189 

time  involved,  formaldehyde  gas,  generated  from  com- 
mercial formalin,  is  not  much  more  expensive  than 
sulphur  dioxide — viz.,  fifteen  to  thirty  cents  per  1000 
cubic  feet  against  ten  cents  with  sulphur.  Therefore, 
we  believe  that  formaldehyde  gas  is  the  best  disinfect- 
ant at  present  known  for  the  surface  disinfection  of 
infected  dwellings.  For  heavy  goods  it  is  far  inferior 
in  penetrative  power  to  steam;  but  for  the  disinfection 
of  fine  wearing  apparel,  furs,  leather,  upholstery,  books, 
and  the  like,  which  are  injured  by  great  heat,  it  is, 
when  properly  employed,  better  adapted  than  any  other 
disinfectant  now  in  use. 

Sulphur  Dioxide  in  House  Disinfection.  Four  pounds 
of  sulphur  should  be  burned  for  every  1000  cubic  feet. 
The  sulphur  should  be  broken  into  small  pieces  and 
put  in  a  pan  sufficiently  large  not  to  allow  the  melted 
sulphur  to  overflow.  This  pan  is  placed  in  a  much 
larger  pan  holding  a  little  water.  The  cracks  of  the 
room  should  be  carefully  pasted  up  and  the  door,  after 
closing,  also  sealed.  Upon  the  broken  sulphur  is  poured 
three  to  four  ounces  of  alcohol  and  the  whole  lighted 
by  a  match.  The  alcohol  is  not  only  for  the  purpose 
of  aiding  the  sulphur  to  ignite,  but  also  to  add  moisture 
to  the  air.  An  exposure  of  eight  to  twelve  hours  should 
be  given. 

Sulphur  fumigation  carried  out  as  above  indicated  is 
not  as  efficient  as  formaldehyde  fumigation,  but  seems 
to  suffice  for  surface  disinfection  for  diphtheria  and  the 
exanthemata.  All  heavy  goods  should  be  removed 
for  steam  disinfection  if  there  is  any  possibility  of  the 
infection  having  penetrated  beneath  their  surface.  If 
there  is  no  place  for  steam  disinfection  their  surfaces 
should  be  thoroughly  exposed  to  fumigation  and  then  to 


190  BACTERIOLOGY. 

the  air  and  sunlight.  In  many  cases  when  cleanliness 
has  been  observed,  surface  disinfection  of  halls,  bedding, 
and  furniture  may  be  all  that  will  be  required. 

There  is  always  a  very  slight  possibility  of  a  deeper 
penetration  of  infection  than  that  believed  to  have 
occurred;  it  is,  therefore,  better  to  be  more  thorough 
than  is  considered  necessary  rather  than  less. 

Sulphur  dioxide  without  the  addition  of  moisture  has, 
as  already  stated  under  the  consideration  of  disinfect- 
fants,  very  little  germicidal  value  upon  dry  bacteria. 

Public  Steam  Disinfecting  Chambers. 

These  should  be  of  sufficient  size  to  receive  all  neces- 
sary goods,  and  may  be  either  cylindrical  or  rectangular 
in  shape,  and  are  provided  with  steam-tight  doors  open- 
ing at  either  end,  so  that  the  goods  put  in  at  one  door 
may  be  removed  at  the  other.  When  large  the  doors 
are  handled  by  convenient  cranes  and  drawn  tight  by 
drop-forged  steel  eye-bolts  swinging  in  and  out  of  slots 
in  the  door  frames.  The  chambers  should  be  able  to 
withstand  a  steam-pressure  of  at  least  one- half  an  at- 
mosphere, and  should  be  constructed  with  an  inside 
jacket,  either  in  the  form  of  an  inner  and  outer  shell  or 
of  a  coil  of  pipes.  This  jacket  is  filled  with  steam  dur- 
ing the  entire  operation,  and  is  so  used  as  to  bring  the 
goods  in  the  disinfecting  chamber  up  to  the  neighbor- 
hood of  220°  F.  before  allowing  the  steam  to  pass 
in.  This  heats  the  goods,  so  that  the  steam  does  not 
condense  on  coming  in  contact  with  them.  It  is  an 
advantage  to  displace  the  air  in  the  chamber  before 
throwing  in  the  steam,  as  hot  air  has  far  less  germicidal 
value  than  steam  of  the  same  temperature.  To  do  this, 
a  vacuum  pump  is  attached  to  the  piping,  whereby  a 


DISINFECTION  AND  STERILIZATION.        191 

vacuum  of  fifteen  inches  can  be  obtained  in  the  cham- 
ber. The  steam  should  be  thrown  into  the  chamber  in 
large  amount,  both  above  and  below  the  goods,  and  the 
excess  should  escape  through  an  opening  in  the  bottom 
of  the  chamber,  so  as  to  more  readily  carry  off  with  it 
any  air  still  remaining.  The  live  steam  in  the  cham- 
ber should  be  under  a  pressure  of  two  to  three  pounds, 
so  as  to  increase  its  action. 

To  disinfect  the  goods,  we  place  them  in  the  chamber, 
close  tight  the  doors,  and  turn  the  steam  into  the  jacket. 
After  about  ten  minutes,  when  the  goods  have  become 
heated,  a  vacuum  of  ten  to  fifteen  inches  is  produced, 
and  then  the  live  steam  is  thrown  in  for  twenty  min- 
utes. The  steam  is  now  turned  off,  a  vacuum  is  again 
formed,  and  the  chamber  again  superheated.  The 
goods  are  now  thoroughly  disinfected  and  dry.  In 
order  to  test  the  thoroughness  of  any  disinfection,  or  any 
new  chamber  maximum,  thermometers  are  placed,  some 
free  in  the  chamber  and  others  surrounded  by  the 
heaviest  goods.  It  will  be  found  that,  even  under  a 
pressure  of  three  pounds,  live  steam  will  require  ten 
minutes  to  penetrate  heavy  goods. 

The  Disinfection  of  Hands,  Instruments,  Ligatures,  and 
Dressings  for  Surgical  Operations. 

Instruments.  All  instruments,  except  knives,  after 
having  been  thoroughly  cleansed,  are  boiled  for  'three 
minutes  in  a  1  per  cent,  solution  of  washing  soda. 
Knives,  after  having  been  thoroughly  cleansed,  are 
washed  in  sterile  alcohol  and  wiped  with  sterile  gauze 
and  then  put  into  boiling  soda  solution  for  one  minute. 
This  will  not  injure  their  edges  to  any  great  extent. 

Gauze.     Gauze  is  sterilized  by  moist  heat  either  in 


192  BACTERIOLOGY. 

an  Arnold  steam  sterilizer  for  one  hour  or  in  an  auto- 
clave for  thirty  minutes.  It  is  placed  in  a  perforated 
cylinder  or  wrapped  in  clean  towels  before  putting  in 
the  sterilizer,  and  only  opened  at  the  operation. 

lodoform  gauze  is  best  made  by  sprinkling  sterile 
iodoform  on  plain  gauze  sterilized  as  described  above. 

Ligatures — Catgut.  Boil  for  one  hour  in  alcohol  under 
pressure  at  about  97°  C.  It  is  often  put  in  sealed  glass 
tubes,  which  are  boiled  under  pressure.  These  remain 
indefinitely  sterile.  The  alcohol  does  not  injure  the 
catgut.  If  desired,  the  catgut  can  be  washed  in  ether 
and  can  be  soaked  a  short  time  in  bichloride  before 
heating  in  alcohol.  Boeckman,  of  St.  Paul,  suggested 
wrapping  the  separate  strands  of  catgut  in  paraffin 
paper  and  then  heating  for  three  hours  at  140°.  This 
procedure  prevents  the  drying  out  of  the  moisture  and 
fat  from  the  catgut,  so  that  it  remains  unshrivelled  and 
flexible  after  its  exposure.  Darling,  of  Boston,  tested 
this  method  and  found  it  satisfactory.  Dry  formalde- 
hyde gas  does  not  penetrate  sufficiently,  and  is  not  reli- 
able. Silver  wire,  silk,  silkworm-gut,  rubber  tubing, 
and  catheters  are  boiled  the  same  as  the  instruments. 

The  Skin  of  the  Patient.  This  is  washed  thoroughly 
with  soap  and  water,  then  with  alcohol,  and  finally  with 
1  :  1000  bichloride.  A  soap  poultice  is  now  placed  on 
for  six  to  twelve  hours,  and  after  its  removal  the  skin 
is  covered  with  a  gauze  compress  previously  moistened 
with  a  1  : 1000  bichloride  of  mercury  solution.  At  the 
operation  the  skin  is  washed  off  with  alcohol  and  then 
with  the  bichloride  of  mercury  solution. 

The  Hands.  Furbinger's  method,  slightly  modified, 
is  now  much  used,  and  gives  good  results :  The  hands 
are  washed  in  hot  soap  and  water  for  five  minutes, 


DISINFECTION  A  ND  STERILIZA  TION.        1  93 

using  the  nail-brush.  They  are  then  soaked  in  alcohol 
for  one  minute  and  scrubbed  with  a  sterile  brush. 
They  are  finally  soaked  in  a  1  :  1000  bichloride  of  mer- 
cury solution  for  three  minutes. 

Sterilized  rubber  gloves  are  now  being  used  more 
and  more  in  operations.  The  gloves  can  be  sterilized 
by  being  left  for  one  minute  in  boiling  1  per  cent,  soda 
solution,  or  they  can  be  sterilized  by  steam. 

The  surgeon's  gowns  and  caps  are  sterilized  by  steam. 
Mucous  membranes,  as  those  of  the  mouth  and  throat, 
are  cleansed  by  a  solution  consisting  of  equal  parts  of 
peroxide  of  hydrogen  and  lime-water.  In  the  nostrils 
it  is  better  to  employ  the  milder  solutions,  such  as 
diluted  DobelPs  or  listerine.  These  are  also  used  in 
the  mouth  instead  of  the  peroxide. 

The  vagina  is  swabbed  out  thoroughly  with  sterile 
warm  soap  and  water  and  then  irrigated  with  a  2  per 
cent,  carbolic  acid  or  a  1  :  1000  bichloride  of  mercury 
solution. 

Hypodermic  syringes  and  other  syringes  are  steril- 
ized by  drawing  up  into  them  boiling  water  a  number 
of  times  and  then  finally  a  5  per  cent,  solution  of  car- 
bolic acid,  the  acid  after  three  minutes  to  be  washed 
out  by  boiling  water.  If  cold  water  is  used  the  car- 
bolic solution  should  remain  in  the  barrel  for  ten  min- 
utes. Great  care  should  be  taken  to  wash  out  all  pos- 
sible matter  before  using  the  carbolic  acid  to  sterilize. 
Syringes  made  entirely  of  glass  or  of  glass  and  asbestos 
can  be  boiled  in  soda  solution. 

THE  STERILIZATION  OF  MILK. 

Bacteria  when  allowed  to  develop  in  milk  produce 
fermentation  (souring)  and  render  the  milk  unfit  to 

13 


194  BACTERIOLOGY. 

be  used  as  an  article  of  food,  especially  for  infants. 
Milk  as  it  reaches  the  city  contains  enormous  num- 
bers of  germs,  and  these  will  produce  fermentation, 
even  though  the  milk  is  kept  on  ice.  Unclean  vessels 
hasten  this  process.  No  matter  how  good  milk  may 
be  in  the  morning,  when  comparatively  fresh,  toward 
evening,  unless  it  has  been  partly  or  completely  steril- 
ized, it  may  be  dangerous  to  an  infant,  and  may,  espe- 
cially in  summer,  cause  fatal  illness,  even  though  it 
still  tastes  sweet. 

Complete  sterilization  destroys  all  the  germs  in  milk, 
and  so  prevents  permanently  fermentative  changes.  By 
partial  sterilization  most  of  the  germs  which  are  not  in 
the  spore  form  may  be  destroyed,  so  that  the  milk  will 
remain  wholesome  for  at  least  twenty -four  hours  in  the 
warmest  weather. 

Milk  is  best  sterilized  by  steam,  for  nearly  all  chem- 
icals, such  as  boric  acid,  salicylic  acid,  and  formalin, 
make  the  milk  less  digestible,  and,  as  a  rule,  unfit  for 
food.  It  may  be  sterilized  at  a  high  or  low  tempera- 
ture— that  is,  at  the  boiling  temperature  (212°  F.) — or 
at  a  lower  degree  of  heat,  obtained  by  modifying  the 
steaming  process. 

It  has  been  found  that  milk  sterilized  at  a  high  tem- 
perature (212°  F.)  is  not  desirable  for  prolonged  use,  as 
the  high  temperature  causes  certain  changes  in  the  milk, 
which  make  it  less  suitable  as  a  food  for  infants.  These 
changes  are  almost  altogether  avoided  if  a  temperature 
below  80°  C.  is  used.  It  is  recommended,  therefore, 
that  the  lowest  temperature  be  used  for  partial  sterili- 
zation, which  will  keep  the  milk  wholesome  for  twenty- 
four  hours  in  the  warmest  weather  and  kill  the  tubercle, 
typhoid,  and  other  non-spore-bearing  bacilli.  Raising 


DISINFECTION  AND  STERILIZATION.        195 

the  milk  to  a  temperature  of  70°  C.  for  fifteen  minutes 
or  80°  C.  for  twelve  minutes  will  accomplish  this. 
One  of  the  many  forms  of  apparatus  is  the  following: 

(a)  A  tin  pail  or  pot,  about  ten  inches  deep  by  nine 
inches  in  diameter,  provided  with  the  ordinary  tin 
cover,  which  has  been  perforated  with  eight  holes,  each 
an  inch  in  diameter. 

(6)  A  wire  basket,  with  eight  nursing  bottles  (as  sold 
in  the  shops  for  this  purpose). 

(c)  Rubber  corks  for  the  bottles  and  a  bristle  brush 
for  cleaning  them. 

Directions  (Koplik).  Place  the  milk,  pure  or  diluted 
(as  the  doctor  may  direct),  in  the  nursing-bottles  and 
place  the  latter  in  the  wire  basket.  Put  only  sufficient 
milk  for  one  nursing  in  each  bottle.  Do  not  cork  the 
bottles  at  first. 

Having  previously  poured  about  two  inches  of  water 
in  the  tin  pail  or  pot  and  brought  it  to  the  boiling- 
point,  lower  the  basket  of  nursing  bottles  slowly  into 
the  pot.  Do  not  allow  the  bottles  to  touch  the  water 
or  they  will  crack.  Put  on  the  perforated  cover  and 
let  the  steaming  continue  for  ten  minutes;  then  remove 
the  cover  and  firmly  cork  each  bottle.  After  replacing 
the  cover,  allow  the  steaming  to  continue  for  fifteen 
minutes.  The  steam  must  be  allowed  to  escape  freely 
or  the  temperature  will  rise  too  high. 

The  process  of  sterilization  is  now  completed.  Place 
the  basket  of  bottles  in  a  cool,  dark  place  or  in  an  ice- 
chest.  The  bottles  must  not  be  opened  until  just  before 
the  milk  is  to  be  used,  and  then  it  may  be  warmed  by 
plunging  the  bottle  in  warm  water.  If  properly 
prepared  the  milk  will  taste  but  little  like  boiled 
milk. 


196  BACTERIOLOGY. 

The  temperature  attained  under  the  conditions  stated 
above  will  not  exceed  in  extreme  cases  188°  F.  (87°  C.). 

Milk  should  be  sterilized  when  it  is  as  fresh  as  pos- 
sible, and  only  sufficient  milk  for  twenty-four  hours 
should  be  sterilized  at  one  time.  If,  after  nursing,  the 
infant  leaves  some  milk  in  the  bottle,  this  should  be 
thrown  away. 

Care  of  the  Bottles  is  Important.  After  nursing,,  the 
bottles  should  be  filled  with  a  strong  solution  of  wash- 
ing soda,  allowed  to  stand  twenty-four  hours,  and  then 
carefully  cleaned  with  a  bristle  (bottle)  brush.  The 
rubber  corks  and  nipples  should  be  boiled  after  using 
in  strong  soda  solution  for  fifteen  minutes  and  then 
rinsed  and  dried. 

After  sterilizing  milk  should  never  be  put  into  unster- 
ilized  bottles,  as  this  will  spoil  it. 

A  different  but  admirable  method  is  the  one  devised 
by  Dr.  Freeman.1  Here  a  pail  is  filled  to  a  certain 
mark  with  water  and  then  placed  on  the  stove  until  the 
water  boils.  It  is  then  removed,  and  immediately  a 
milk-holder,  consisting  of  a  series  of  zinc  cylinders,  is 
lowered  with  its  milk  bottles  partially  full  of  milk. 
The  cover  is  again  applied.  The  heat  of  the  outside 
water  raises  the  temperature  of  the  milk  in  ten  minutes 
to  75°  C.  (167°  F.),  and  holds  it  nearly  at  that  point 
for  some  time.2  After  twenty  minutes  the  milk  is  re- 
moved, placed  in  cold  water,  and  quickly  cooled.  The 
milk  is  kept  in  the  ice-chest  until  used. 

1  Agent  for  Pasteurizer,  James  Dougherty,  411  W.  59th  St. 

2  A  temperature  of  75°  C.  is  advised  in  Pasteurizing  milk,  instead  of  65°  C., 
which  would  ordinarily  suffice  to  kill  all  bacteria  free  of  spores,  because  of 
the  fact  pointed  out  by  Theobald  Smith,  that  the  bacteria  embedded  in  the 
pellicle  which  forms  on  the  surface  are  more  resistant  than  those  surrounded 
by  fluid. 


CHAPTER   XII. 

THE    PREPARATION,    STAINING,    AND    MICROSCOPICAL 
EXAMINATION   OF   BACTERIA. 

As  the  purpose  of  this  book  is  to  give,  outside  of 
special  methods  devised  for  purposes  of  diagnosis  and 
the  development  of  curative  serums,  only  such  descrip- 
tions of  technique  as  are  necessary  to  students  in  their 
laboratory  courses,  or  to  physicians  in  the  very  simple 
examinations  which  they  will  be  able  to  carry  on  in 
their  private  offices,  readers  are  referred  to  works 
such  as  Sternberg's  or  Abbott's  for  fuller  descriptions 
of  the  apparatus  and  technique  used  in  bacteriological 
research. 

Since  bacteria  are  present  to  a  greater  or  less  extent 
in  the  air,  earth,  and  water  around  us,  on  our  bodies, 
clothes,  and  all  surrounding  objects,  it  follows  that 
when  we  begin  to  examine  substances  for  bacteria  the 
first  requisite  is  that  all  the  materials  we  use  must  be 
free  and  kept  free  from  bacteria,  both  living  and  dead, 
otherwise  we  cannot  tell  whether  those  we  detect  are 
in  the  substances  examined  or  only  in  the  materials  we 
have  used  in  the  investigation. 

Additional  care  has  to  be  taken  when  we  study  in- 
fection in  the  living  body,  for  in  the  skin  and  mucous 
membranes  there  are  not  only  abundant  bacteria  but 
varieties  similar  to  those  which  produce  disease,  so  that 
if  we  do  not  use  the  greatest  precautions  we  will  con- 
taminate our  material  with  these  bacteria  and  get  utterly 


198  BACTERIOLOGY. 

misleading  results.  After  death  we  have  an  added  dif- 
ficulty, in  that  even  the  blood  and  body  tissues  become 
invaded  by  bacteria  from  the  intestines  and  elsewhere, 
so  that  bacteria  actually  present  in  the  diseased  tissues 
may  have  had  no  connection  with  the  disease  under  in- 
vestigation. Whenever  bacteria  are  found,  therefore, 
the  methods  carried  out  in  the  investigation  should  be 
most  carefully  examined,  to  see  if  some  error  in  tech- 
nique has  not  been  committed.  The  aim  of  the 
bacteriological  examination  of  any  material  is  to  de- 
termine whether  bacteria  are  present  or  not,  and,  if 
present,  to  ascertain  their  number  and  distribution,  and, 
if  possible,  their  species.  This  is  accomplished  chiefly 
by  means  of  two  methods — viz.,  the  direct  examina- 
tion with  the  microscope  of  cover  glass  preparations 
and  the  results  of  cultures  made  from  the  material. 
Sometimes  animal  inoculations  are  also  employed. 

The  direct  microscopical  examination  of  suspected  sub- 
stances for  bacteria  can  be  made  either  with  or  without 
staining.  Unstained,  the  bacteria  are  examined,  to  note 
their  motility,  their  form, and  their  general  arrangement: 
but  for  more  exact  study,  they  can  be  so  much  better 
observed  when  stained  that  this  step  is  always  advisable. 
A  cover-glass  preparation  is  made  as  follows :  A 
very  small  amount  of  the  blood,  pus,  discharges  from 
mucous  membranes,  culture  fluids,  or  other  material  to 
be  examined  is  removed  by  means  of  a  sterile  swab  or 
platinum  loop  and  smeared  undiluted  in  a  thin  film 
over  a  clean,  thin  cover-glass.1  From  cultures  on  solid 

1  To  render  new  cover-slips  clean  and  free  from  grease,  place  them  in  strong 
nitric  acid  for  a  few  hours,  then  rinse  them  olF  in  water,  then  in  alcohol, 
then  in  ether.  Place  them  finally  for  keeping  in  alcohol,  to  which  a  little 
ammonia  has  been  added.  When  used  wipe  with  soft  clean  handkerchief. 
If  old  cover-slips  are  used  boil  first  in  soda  solution. 


MICROSCOPICAL  EXAMINATION.  199 

media,  however,  on  account  of  the  abundance  of  bacteria 
in  the  material,  a  little  of  the  growth  is  diluted  by  add- 
ing it  to  a  tiny  drop  of  clean  water  which  has  been 
previously  placed  on  the  cover-glass.  The  amount  of 
dilution  is  learned  after  a  few  trials.  It  is  best  to  add 
to  the  drop  just  enough  to  make  a  perceptible  cloudi- 
ness. The  mixture  is  then  smeared  over  the  cover- 
glass.  From  whatever  source  derived,  the  film  is  al- 
lowed to  dry  at  the  usual  air  temperature,  and  then,  in 
order  to  fix  the  film  with  its  contained  bacteria  to  the 
glass,  the  latter  is  passed  three  times  by  a  rather  slow 
movement  through  the  Bunsen  or  alcohol  flame.  In- 
stead of  this  method,  the  film  may  be  fixed  to  the  glass 
by  placing  it  in  absolute  alcohol  for  a  few  minutes. 
The  smear  thus  prepared  is  usually  stained  either 
by  the  simple  addition  of  a  solution  of  an  aniline  dye, 
for  from  one  to  five  minutes,  or  by  one  of  the  more 
complicated  special  stains  described  later,  such  as  that 
of  Gram  or  that  used  for  the  tubercle  bacillus,  where 
the  ability  of  the  bacteria  to  retain  their  stain  when 
placed  in  decolorizing  solutions  is  tested.  When  the 
stain  is  to  be  hastened  or  made  more  intense  the  dye  is 
used  warm.  For  ordinary  staining  the  bacteria  are 
simply  covered  completely  by  the  cold  staining  fluid. 
The  cover-glass,  with  the  charged  side  uppermost,  may 
either  rest  on  the  table  or  be  held  by  some  modifica- 
tion of  Cornet's  forceps.  When  the  solution  is  to  be 
warmed  the  cover-glass  may  be  floated,  smeared  side 
down,  upon  the  fluid  contained  in  a  porcelain  dish  rest- 
ing on  a  wire  mat,  supported  on  a  stand,  or  it  may  be 
held  in  the  Cornet  forceps.  The  fluid  in  both  the  dish 
and  on  the  cover-glass  should  be  carefully  warmed,  so 
as  to  steam  without  actually  boiling.  The  cover-glass 


200  BACTERIOLOGY. 

should  be  kept  completely  covered  with  fluid.  The 
bacteria  having  now  been  stained,  the  cover-glass  is 
grasped  in  the  forceps  and  thoroughly  washed  in  clean 
water  and  then  dried,  first  between  layers  of  filter-paper 
and  then  in  the  air.  A  drop  of  balsam  or  water  is  now 
placed  on  a  glass  slide  and  the  cover-glass  placed  upon 
it  with  the  bacterial  side  down.  The  preparation  is 
now  ready  for  microscopical  examination. 

The  Preparation  of  Sections.  Occasionally  it  is  of  value 
to  examine  the  bacteria  as  they  are  in  the  tissues  them- 
selves. These  should  be  obtained  as  soon  as  possible 
after  death,  so  as  to  prevent  any  post-mortem  changes 
or  increase  of  the  bacteria  in  them.  From  the  properly 
selected  spots  small  portions,  not  larger  than  one  cubic 
centimetre,  are  removed  and  placed  in  absolute  alcohol 
to  harden.  These  portions,  when  of  nearly  the  con- 
sistency of  fresh,  solid  rubber,  are  removed,  and,  if  the 
nature  of  the  tissues  will  allow,  fastened  to  corks  or 
pieces  of  hard  rubber  by  mucilage.  After  dry  ing,  the 
specimens  are  replaced  in  alcohol  for  twenty-four  hours 
and  then  cut  into  thin  sections  with  the  microtome. 
Sometimes  the  tissues  do  not  become  sufficiently  dense 
by  this  simple  method;  they  must  then  undergo  the 
process  of  embedding  in  celloidin  or  paraffin. 

The  Ordinary  Staining  Solutions.  Simple  staining  is 
used  for  the  demonstration  of  bacteria  in  general,  and 
is  also  useful  for  gaining  an  idea  of  the  other  elements  in 
the  preparation.  The  solutions  commonly  employed  in 
staining  bacteria  are  the  watery  solutions  of  basic  aniline 
dyes  —  fuchsin,  gentian-violet,  and  methylene  -  blue. 
These  solutions  may  either  be  prepared  by  dissolving 
the  dyes  directly  in  water,  or,  more  usually,  by  having 
stock  saturated  alcoholic  solutions  of  them,  from  which 


MICROSCOPICAL  EXAMINATION.  201 

we  can  take  from  time  to  time  the  amount  necessary  to 
make  up  the  watery  solutions  for  use.  The  stock  satu- 
rated alcoholic  solutions  are  made  by  pouring  into  a  bot- 
tle enough  of  the  dye  in  substance  to  fill  them  to  about 
one-quarter  of  their  capacity.  The  bottle  should  then 
be  filled  with  alcohol,  tightly  corked,  well  shaken,  and 
allowed  to  stand  for  twenty-four  hours.  If  at  the  end 
of  this  time  all  the  staining  material  has  been  dis- 
solved, more  should  be  added,  the  bottle  being  again 
shaken  and  allowed  to  stand  for  another  twenty-four 
hours.  This  must  be  repeated  until  a  permanent  sedi- 
ment of  undissolved  coloring-matter  is  seen  upon  the 
bottom  of  the  bottle.  This  will  then  be  labelled  t(  satu- 
rated alcoholic  solution,77  of  whatever  dye  has  been 
employed.  The  alcoholic  solutions  are  not  themselves 
employed  for  staining  purposes.  The  solution  for  use 
is  made  by  filling  a  small  bottle  three-fourths  with  dis- 
tilled water,  and  then  adding  the  concentrated  alcoholic 
solution  of  the  dye,  little  by  little,  until  one  can  just 
see  through  the  solution.  Care  must  be  taken  that  the 
color  does  not  become  too  dense.  Small  wooden  cases 
come  prepared  for  holding  about  one-half  dozen  bottles 
of  the  staining  solutions.  This  number  will  answer 
for  all  routine  purposes  of  the  student  or  physician. 

For  certain  bacteria,  which  stain  only  imperfectly 
with  these  solutions,  it  is  necessary  to  employ  some 
agent  that  will  increase  the  penetrating  action  of  the 
dyes.  We  have  learned  that  the  addition  to  a  solu- 
tion of  a  small  quantity  of  alkaline  substance,  or  by 
dissolving  the  staining  materials  in  strong  watery  solu- 
tions of  either  aniline  oil  or  carbolic  acid,  instead  of 
simple  water,  will  accomplish  this.  Of  the  solutions 
thus  prepared  there  are  three  in  common  use:  Loeffler's 


202  BACTERIOLOGY. 

alkaline  methylene-blue  solution,  the  Koch-Ehrlich 
aniline  water  solution,  of  either  fuchsin,  gentian-violet, 
or  methylene-blue,  and  Ziehl's  solution  of  fuchsin  in 
carbolic  acid.  These  solutions  are  as  follows  : 

Loeffler's  Alkaline  Methylene-blue  Solution.  This  con- 
sists of  concentrated  alcoholic  solution  of  methylene- 
blue,  30  c.c.;  caustic  potash  in  one  ten  thousandth 
solution,  100  c.c. 

Koch-Ehrlich  Aniline  Water  Solution  of  Fuchsin  or 
Gentian-violet  is  prepared  as  follows:  To  about  100 
c.c.  of  distilled  water,  aniline  oil  is  added,  drop  by 
drop,  until  it  has  an  opaque  appearance,  the  solution 
being  thoroughly  shaken  after  the  addition  of  each  drop. 
It  is  then  filtered  into  a  beaker  through  moistened 
filter-paper  until  the  filtrate  is  perfectly  clear.  To 
100  c.c.  of  the  filtrate  add  10  c.c.  of  absolute  alcohol 
and  11  c.c.  of  the  concentrated  alcoholic  solution  of 
either  fuchsin,  methylene-blue,  or  gentian- violet. 

Ziehl's  Carbolic  Fuchsin  Solution.  Distilled  water, 
100  cc. ;  carbolic  acid  (crystalline),  5  grams;  absolute 
alcohol,  10  c.c  ;  fuchsin,  1  gram;  or  it  may  be  pre- 
pared by  adding  to  a  5  per  cent,  watery  solution  of 
carbolic  acid  the  saturated  alcoholic  solution  of  fuchsin 
until  a  metallic  lustre  appears  on  the  surface  of  the 
fluid. 

The  last  two  methods,  combined  with  heating,  are 
used  to  stain  the  bacteria  intensely,  so  that  the  more 
resistant  of  them  may  retain  their  color  when  exposed 
to  decolorizing  agents.  When  so  treated  certain  of 
the  bacteria  will  retain  their  color,  even  when  exposed 
to  very  strong  decolorizers.  The  bacilli  of  tuberculosis 
and  of  leprosy  are  examples.  They  are  both  difficult 
to  stain,  but  when  once  stained  are  equally  resistant  to 


MICROSCOPICAL  EXAMINATION.  203 

give  up  their  stain.  The  details  of  staining  tubercle 
bacilli  will  be  found  under  Tuberculosis. 

Another  differential  method  of  staining  which  is 
very  commonly  employed  is  that  known  as  Gram's 
method.  In  this  method  the  objects  to  be  stained 
are  covered  with  the  aniline  gentian- violet  solution. 
After  remaining  in  this  for  a  few  minutes  they  are 
immersed  in  an  iodine  solution,  composed  of  iodine,  1 
grain;  potassium  iodide,  2  grains;  distilled  water,  300 
c.c.  In  this  they  remain  for  from  one  to  two  minutes. 
They  are  then  transferred  to  alcohol  and  thoroughly 
rinsed.  If  the  cover-glass  as  a  whole  still  shows  a 
violet  color,  it  is  again  treated  with  the  iodine  solu- 
tion, followed  by  alcohol,  and  this  is  continued  until 
no  trace  of  violet  color  is  visible  to  the  naked  eye. 
They  may  then  be  washed  in  water  and  examined,  or 
a  contrasting  color  of  carmine  or  Bismarck  brown  may 
be  given  them.  This  method  is  particularly  useful  in 
demonstrating  the  capsule  which  is  seen  to  surround 
some  bacteria  —  particularly  the  pneumococcus  —  and 
also  in  differentiating  between  varieties  of  bacteria,  for 
some  do  and  others  do  not  retain  their  stain  when  put 
in  the  iodine  solution  for  a  suitable  time. 

Another  method  of  demonstrating  the  capsule  is  the 
glacial  acetic  acid  method,  as  described  by  Welch: 
1.  Cover  the  preparation  with  glacial  acetic  acid  for  a 
few  seconds.  2.  Drain  off  and  replace  with  aniline 
gentian-violet  solution;  this  is  to  be  repeatedly  added 
until  all  the  acid  is  replaced.  3.  Wash  in  2  per  cent, 
solution  of  sodium  chloride  and  mount  in  the  same. 

Staining  the  Spores.  We  have  already  noted  that 
during  certain  stages  in  the  growth  of  a  number  of 
bacteria  spores  are  formed  which  refuse  to  take  up  color 


204  BA  CTERIOL  OGY. 

when  the  bacteria  are  stained  in  the  ordinary  manner. 
Special  stains  have  been  devised  for  causing  the  color  to 
penetrate  through  the  resistant  spore.  Thus  in  Abbott's 
method  the  cover-slip  after  having  been  prepared  in  the 
usual  way  is  covered  with  a  dye  and  held  over  the  Bun- 
sen  flame  until  the  fluid  steams.  This  is  continued  for 
one  or  two  minutes.  It  is  then  washed  and  dipped  in  a 
decolorizing  acid  solution,  such  as  a  2  per  cent,  alco- 
holic solution  of  nitric  acid,  until  all  visible  color  has 
disappeared,  then  it  is  washed  off  and  dipped  for  ten  sec- 
onds in  a  solution  containing  10  parts  saturated  alco- 
holic solution  of  eosin  and  90  of  water.  The  bacilli 
will  then  be  rose-colored  and  the  spores  blue.  Some- 
times, however,  the  spores  refuse  to  take  the  stain  in 
this  manner.  We  then  can  adopt  Moeller's  method, 
which  is  designed  still  further  to  favor  the  penetration 
of  the  coloring-matter  through  the  spore  membrane. 
He  macerates  the  spores  in  a  solution  of  chromic  acid 
before  staining  them.  The  prepared  cover-slip  is  held 
for  two  minutes  in  chloroform,  then  washed  off  in  water, 
then  placed  from  one-half  to  three  minutes  in  a  5  per 
cent,  solution  of  chromic  acid,  again  washed  off  in 
water,  and  now  restained  by  adding  to  it  carbolic 
fuchsin,  which  is  steamed  for  several  minutes.  The 
staining  fluid  is  then  washed  off  and  the  preparation 
decolorized  in  a  3  per  cent,  solution  of  hydrochloric 
acid  or  a  5  per  cent,  solution  of  sulphuric  acid.  The 
preparation  is  finally  stained  for  a  minute  in  methyleue- 
blue  solution.  The  spores  will  be  red  and  the  body  of 
the  cells  blue.  The  different  spores  vary  greatly  in 
the  readiness  with  which  they  take  up  the  dyes,  and 
we  have,  therefore,  to  experiment  with  each  variety 
as  to  the  length  of  time  they  should  be  exposed  to  the 


MICROSCOPICAL  EXAMINATION.  205 

maceration  of  the  chromic  acid.  Even  under  the  best 
conditions  it  is  almost  impossible  to  stain  some  spores. 

Staining  Flagella.  For  the  demonstration  of  flagella, 
which  are  possessed  by  all  motile  bacteria,  we  are  in- 
debted to  Loeffler.  The  special  stains  devised  by  him, 
and  also  the  one  devised  by  Van  Ermengem,  are  those 
usually  employed. 

Bunge's  modification  of  Loeffler7 s  method  is  carried 
out  as  follows:  Cover-glasses  which  have  been  most 
carefully  cleaned  are  covered  by  a  very  thin  smear  of 
an  eighteen-hours'  old  culture  of  the  motile  organism 
to  be  examined.  After  drying  in  the  air  and  passing 
three  times  through  the  flame  the  smear  is  treated  with 
a  mordant  solution,  which  is  prepared  as  follows:  To 
3  parts  of  saturated  alum  solution  add  1  part  of  a  solu- 
tion of  liquor  ferri  sesquichloride,  of  the  strength  of 
1:20  of  distilled  water.  To  10  c.c.  of  this  mixture 
add  1  c.c.  of  a  concentrated  watery  solution  of  fuchsin. 
This  mordant  should  be  allowed  to  stand  for  several 
days  before  using.  After  preparing  the  cover-slip  with 
all  precautions  necessary  to  cleanliness  the  filtered  mor- 
dant is  allowed  to  act  cold  for  five  minutes,  after  which 
it  is  slightly  warmed  and  then  washed  off.  After  dry- 
ing the  smear  is  faintly  stained  with  the  carbol  fuchsin 
solution  and  then  washed  off,  dried,  and  mounted. 

Frequently  the  flagella  appear  well  stained,  but  often 
the  process  has  to  be  repeated  a  number  of  times  before 
success  is  arrived  at. 

The  Preservation  of  Specimens.  Dry  unstained  or 
stained  preparations  of  bacteria  keep  indefinitely  if 
mounted  in  Canada  balsam,  cedar  oil,  or  dammar  lac; 
they  tend  to  gradually  fade,  but  may  be  preserved  for 
many  months  or  years. 


206 


BACTERIOLOGY. 


The  Microscopical  Examination  of  Bacteria. 

The  Different  Parts  of  the  Microscope.  A  complete 
instrument  usually  has  four  oculars,  or  eye-pieces  (A), 
which  are  numbered  from  1  to  4,  according  to  the 


FIG.  16. 


— G 


Microscope. 


MICROSCOPICAL  EXAMINATION.  207 

amount  of  magnification  which  they  yield.  Numbers 
2  and  4  are  most  useful  for  bacteriological  work.  The 
objective  (B) — the  lens  at  the  distal  end  of  the  barrel — 
serves  to  give  the  main  magnification  of  the  object.  For 
stained  bacteria  the  1/12  achromatic  oil  immersion  lens 
is  regularly  employed;  except  for  photographic  purposes 
the  apochromatic  lenses  are  not  needed.  Even  here 
they  are  not  indispensable.  A  1/10  or  1/16  may  at 
times  be  useful  but  hardly  necessary;  a  No.  4  ocular  and 
a  1/12  lens  give  a  magnification  of  about  1000  diame- 
ters. (Fig.  17.)  For  unstained  bacteria  we  employ 

FIG.  17. 


Anthrax  bacilli  and  blood-cells.    X  1000  diameters. 

either  the  1/12  immersion  or  1/7  dry  lens,  according  to 
the  purpose  for  which  we  study  the  bacteria;  for  the 
examination  of  colonies  where,  as  a  rule,  we  do  not 
wish  to  see  individual  bacteria  but  only  the  general 
appearance  of  whole  groups,  we  use  lenses  of  much 
lower  magnification.  (Fig.  18.) 

The  stage  (C) — the  platform  upon  which  the  .object 
rests — should   be  large   enough   to  support  the  Petri 


208  BACTERIOLOGY. 

plates  if  culture  work  is  to  be  done.  The  iris  dia- 
phragm (D),  which  is  now  regularly  used  in  bacterio- 
logical work,  opens  and  closes  like  the  iris  of  the  eye, 
and  so  controls  the  amount  of  light.  Its  opening  is 
diminished  or  increased  by  moving  a  small  arm,  which 
is  underneath  the  stage,  in  one  or  another  direction. 
The  reflector  placed  beneath  the  stage  serves  to  direct 
the  light  to  the  object  to  be  examined.  It  has  two  sur- 
faces— one  concave  and  one  convex.  The  coarse  ad- 

FIG.  18. 


Colonies  of  diphtheria  bacilli.    X  200  diameters. 

justment  (F)  is  the  rack-and-pinion  arrangement  by 
which  the  barrel  of  the  microscope  can  be  quickly 
raised  or  lowered.  It  is  used  to  bring  the  bacteria 
roughly  into  focus.  The  fine  adjustment  (G)  serves  to 
raise  and  lower  the  barrel  very  slowly  and  evenly,  and 
is  used  for  the  exact  study  of  the  bacteria  when  high- 
power  lenses  are  used.  For  the  microscopical  study  of 
bacteria  it  is  essential  that  we  magnify  the  bacteria  as 
much  as  possible  and  still  have  their  definition  clear  and 


MICROSCOPICAL  EXAMINATION.  209 

sharp.  It  is  essential,  therefore,  that  the  microscope  be 
provided  with  an  oil  immersion  system  and  a  substage 
condensing  apparatus.  In  using  the  oil  immersion  lens 
a  drop  of  oil  of  the  same  index  of  refraction  as  the 
glass  is  placed  upon  the  face  of  the  lens,  so  as  to  con- 
nect it  with  the  cover-glass  when  the  bacteria  are  in 
focus.  There  is  thus  no  loss  of  light  through  deflection, 
as  is  the  case  in  the  dry  system. 

The  Substage  Condensing  Apparatus  (H)  is  a  system  of 
lenses  situated  beneath  the  central  opening  of  the  stage. 
It  serves  to  condense  the  light  passing  through  the  re- 
flector to  the  object  in  such  a  way  that  it  is  focussed 
upon  the  object,  thus  furnishing  the  greatest  amount  of 
luminosity.  Between  the  condenser  and  the  reflector  is 
placed  an  adjustable  diaphragm,  the  aperture  of  which 
can  be  regulated,  as  circumstances  require,  to  permit 
of  either  a  very  small  or  a  very  large  amount  of  light 
passing  to  the  object. 

The  Examination  of  Bacteria  in  the  Hanging  Drop. 
It  is  often  valuable  to  observe  bacteria  alive,  so  as  to 
study  them  under  natural  conditions.  We  can  thus 
note  the  method  and  rate  of  their  multiplication, 

FIG.  19. 


Hollow  slide  with  cover-glass. 

whether  they  move  and  produce  spores,  and  whether 
or  not  they  clump  or  disintegrate  with  specific  serums. 
For  this  special  slides  and  methods  are  desirable.  The 
usual  form  is  one  in  which  there  is  ground  out  on  one 
surface  a  hollow  having  a  diameter  of  about  half  an 
inch.  (Fig.  19.)  According  to  the  purpose  for  which 

14 


210  BACTERIOLOGY. 

the  hanging  drop  is  to  be  studied,  sterilization  of  the 
slide  and  cover-glass  may  or  may  not  be  necessary. 
The  technique  of  preparing  and  studying  the  hanging 
drop  is  as  follows  :  The  surface  of  the  glass  around  the 
hollow  in  the  slide  is  smeared  with  a  little  vaseline  or 
other  inert  substance.  This  has  for  its  purpose  both 
the  sticking  of  the  cover-glass  to  the  slide  and  the  pre- 
vention of  evaporation  in  the  drop  placed  in  the  little 
chamber,  which  is  to  be  formed  between  the  cover- 
glass,  when  placed  over  the  hollow,  and  the  slide. 
For  the  purpose  of  studying  the  bacteria  we  place,  if 
they  are  in  fluids,  simply  a  platinum  loopful  upou  the 
centre  of  the  cover-glass  and  then  invert  it  by  means  of 
a  slender  pair  of  forceps  over  the  hollow  in  the  slide, 
being  very  careful  to  have  the  bacteria  over  the  very 
centre  of  the  space.  If  the  bacteria,  on  the  contrary, 
are  growing  on  solid  media,  or  are  obtained  from  thick 
pus  or  tissues  from  organs,  they  are  mixed  with  a  suit- 
able amount  of  bouillon  or  sterile  physiological  salt 
solution  either  before  or  after  being  placed  upon  the 
cover  glass.  If  we  wish  to  observe  the  bacteria  under 
natural  conditions  we  must  keep  the  tiny  drop  of  fluid 
at  the  proper  temperature  for  the  best  growth  of  the 
bacteria.  If,  however,  we  simply  wish  to  observe  their 
form  and  arrangement  this  is  not  necessary.  In  the 
study  of  living  bacteria  we  often  wish  to  observe  their 
grouping  and  motion  rather  than  their  individual  char- 
acters, and  so  use  less  magnification  than  for  stained 
bacteria.  In  studying  unstained  bacteria  and  tissues 
we  shut  off  as  large  a  portion  of  the  light  with  our 
diaphragm  as  is  compatible  with  distinct  vision,  and 
thus  favor  contrasts  which  appear  as  lights  and  shadows, 
due  to  the  differences  in  light  transmission  of  the  dif- 


MICROSCOPICAL  EXAMINATION.  211 

ferent  materials  under  examination.  It  is  necessary 
to  remember  that  they  are  seen  with  difficulty,  and  that 
we  are  very  apt,  unless  extremely  careful  in  focussing, 
to  allow  the  lens  to  go  too  far,  and  so  come  upon  the 
cover-glass,  break  it,  destroy  our  preparation,  and,  if 
examining  parasitic  bacteria,  infect  the  lens.  This  may 
be  avoided  by  first  finding  the  hanging  drop  with  a  low 
power  lens  and  thus  exactly  centre  it.  The  lens  of 
higher  magnification  is  now  very  gradually  lowered, 
while  at  the  same  time  gently  moving  the  slide  back 
and  forth  to  the  slightest  extent  possible  with  the  left 
hand.  If  any  resistance  is  felt  raise  the  lens,  for  it  has 
gone  beyond  the  point  of  focus  and  is  touching  the 
cover-glass. 


CHAPTER  XIII. 

BACTERIOLOGICAL   TECHNIQUE — Continued. 

THE  CULTIVATION   OF   BACTERIA. 

IN  order  to  determine  the  number  of  living  bacteria 
in  any  substance  and  their  nature  we  have  to  cultivate 
and  isolate  them. 

The  Most  Common  of  the  Nutrient  Media  Used  for  the 
Growth  of  Bacteria. 

All  of  these  must  have,  as  noted  earlier,  food  con- 
taining the  necessary  carbon,  nitrogen,  and  mineral 
substances  in  a  form  easily  assimilated  and  in  the 
proper  concentration.  The  pathogenic  bacteria  nearly 
all  require  for  good  growth  peptone,  albumins,  and 
sugar.  For  each  kind  the  proper  food  must  be  found 
through  experimentation,  as  slight  alterations  may 
make  a  great  difference. 

Physicians  will  find  it,  as  a  rule,  convenient  to 
purchase  their  media  already  prepared  from  some  of 
the  reliable  firms  that  deal  in  bacteriological  products. 
Special  media,  such  as  those  employed  for  isolation  and 
identification  of  the  typhoid  bacillus  and  gonococcus, 
will  be  found  described  along  with  those  bacteria.  For 
those  who  may  wish  to  make  their  own,  we  will  de- 
scribe here  those  in  common  use: 

Nutrient  Bouillon  or  Broth.  One  part  of  finely  chopped 
fresh,  lean  meat  is  macerated  in  two  parts  of  water  and 


BACTERIOLOGICAL  TECHNIQUE. 


213 


put  in  an  ice-chest  for  from  eighteen  to  twenty-four 
hours.  The  infusion  is  strained,  when  cold,  through  a 
fine  cheese-cloth,  and  to  the  clear  filtrate  1  per  cent,  of 
peptone  and  0.5  per  cent,  of  sodium  chloride  are  added. 
The  medium  is  then  warmed  for  some  minutes  until  the 
peptone  is  dissolved,  and  then  exposed  to  live  steam 
either  without  pressure  in  the  Arnold  steam  sterilizer 
(Fig.  20)  for  thirty  minutes,  or  in  the  autoclave  (Fig. 


FTG.  20. 


STERILIZING  CHAMBER 

U  A 


Arnold  steam  sterilizer. 

21)  at  one  atmosphere  of  pressure  for  fifteen  minutes, 
or  boiled  over  a  free  flame  for  ten  minutes.  While 
still  hot  it  is  filtered  through  filter-paper  or  through 
absorbent  cotton,  and  the  reaction  is  tested  and  suf- 
ficient hydrochloric  acid  or  sodium  hydroxide  added 


214 


BACTERIOLOGY. 


to  give  it  the  desired  reaction,  which  is  for  most  bac- 
teria slightly  alkaline  to  litmus.  If  the  fluid  is  clear 
it  is  put  into  flasks  and  tubes  and  sterilized;  if  not 
clear,  the  white  of  one  or  two  eggs  is  added  to  the 
fluid  after  cooling  it  down  to  about  55°  C.  After 


FIG.  21. 


Autoclave  for  sterilization  with  live  steam  under  pressure. 

thoroughly  mixing  the  eggs  the  bouillon  is  boiled 
briskly  for  a  few  minutes  and  then  again  filtered  and 
distributed  in  flasks  and  tubes  and  put  in  the  Arnold 
sterilizer  for  one  hour  on  each  of  two  consecutive  days, 
or  in  the  autoclave  for  twenty  minutes  for  sterilization. 
Instead  of  meat  2  to  4  grammes  of  Liebig's  or  some 
other  meat  extract  are  added  to  each  litre  of  water. 
For  most  purposes  the  extract  is  as  good  as  the  fresh 
meat,  but  for  toxin  production  it  is  inferior. 


BACTERIOLOGICAL  TECHNIQUE.  215 

Fermentation  broth  is  made  usually  by  adding  1  per 
cent,  of  glucose  to  the  above.  For  accurate  work  the 
meat  sugars  are  first  extracted  by  allowing  the  colon 
bacillus  to  grow  in  the  broth  over  night.  The  bouillon 
is  then  sterilized  and  the  peptone  and  salt  added,  and 
the  process  already  given  gone  through  with. 

Fermentation  bouillon  is  usually  placed  in  a  tube  of 
special  construction,  known  as  a  fermentation  tube  (see 
Fig.  14,  p.  82).  This  is  essentially  a  tube  1.5  cm.  in 
diameter,  bent  at  an  acute  angle,  closed  at  one  end,  and 
provided  with  a  bulb  at  the  other  end,  which  latter 
should  be  large  enough  to  receive  all  the  fluid  in  the 
closed  branch  should  gas  in  any  considerable  quantity 
collect  there.  The  tube  also  serves  a  most  important  end 
in  giving  information  as  to  the  aerobic  and  anaerobic 
growth  of  the  species  under  consideration,  for  the  con- 
necting tube  being  constricted  serves  to  prevent,  to  a 
great  degree,  the  entrance  of  oxygen  of  the  air  into  the 
closed  branch,  and  the  free  oxygen  in  the  medium  is 
driven  out  by  the  heat  during  sterilization;  from  which 
it  may  be  seen  that  growth  in  the  bulb  is  aerobic  and 
growth  in  the  closed  branch  is  anaerobic.  For  the  study 
of  fermentation  alone  small  tubes  may  be  inverted  into 
larger  ones  or  tubes  may  be  bent  on  themselves. 

Nutrient  Gelatin.  To  the  bouillon  already  prepared 
as  described  add  10  per  cent,  of  sheet  gelatin  and  neu- 
tralize. Add  the  whites  of  two  eggs  for  each  litre  and 
boil  for  a  few  minutes.  Filter,  place  in  tubes  or  flasks, 
and  sterilize.  Instead  of  adding  gelatin  to  bouillon 
already  prepared,  it  may  be  added  to  the  meat  infusion 
at  the  same  time  the  peptone  and  salt  were  added  in 
preparing  nutrient  bouillon  as  just  described. 

Nutrient  Agar.     This  is  prepared  by  adding  to  stock 


216  BACTERIOLOGY. 

bouillon  1  or  2  per  cent.,  as  desired,  of  thread  agar, 
melting  it  by  placing  over  a  free  flame  or  in  the  auto- 
clave or  steam  sterilizer.  When  the  agar  is  brought 
into  solution  over  a  free  flame  there  may  be  consider- 
able loss  of  fluid  by  evaporation.  This  should  be  com- 
pensated for  by  adding  additional  water  before  boiling. 
Agar  may  be  added  directly  to  the  meat  infusion  along 
with  the  peptone  and  salt.  Indeed,  this  is  an  advan- 
tage, as  agar-agar  is  very  difficult  to  bring  into  solution, 
and  is  not  injured  in  the  least  by  prolonged  boiling. 
Glycerin  agar  is  simply  nutrient  agar  plus  3  to  6  per 
cent,  of  glycerin.  It  is  added  to  the  hot  nutrient  agar 
just  previous  to  putting  it  in  the  flasks.  Nutrient  agar 
begins  to  thicken  at  a  fairly  high  temperature,  and 
should  be  filtered  as  hot  as  possible.  When  small 
amounts  are  made  it  is  well  to  place  the  filter  and  re- 
ceiving-flask in  the  sterilizer  while  filtering. 

Milk.  This  fluid  is  a  good  culture  medium  for  most 
pathogenic  bacteria.  It  should  be  obtained  as  fresh  as 
possible,  so  that  but  little  bacterial  change  has  occurred. 
It  is  first  put  in  a  steam  sterilizer  for  fifteen  minutes 
and  then  put  in  the  ice-chest  for  twelve  hours,  to  allow 
the  cream  to  rise.  The  milk  is  then  siphoned  off  from 
below  the  cream  into  a  flask  and  its  reaction  tested.  A  f  ter 
correction  it  is  put  in  tubes  or  flasks  and  sterilized. 

Potatoes.  Potatoes  are  used  for  some  special  pur- 
poses. The  potatoes  may  after  thorough  scrubbing  and 
removal  of  "eyes"  be  soaked  in  bichloride  of  mer- 
cury (1  : 1000)  for  twenty  minutes,  and  then  sterilized 
on  three  consecutive  days  for  one-half  hour  in  the 
steam  sterilizer.  To  use  they  are  cut  in  thick  slices  and 
put  in  deep  Petri  dishes.  For  more  careful  work  the 
potatoes  are  first  cut  into  proper  sizes  for  tubes  or 


BACTERIOLOGICAL  TECHNIQUE.  217 

dishes,  and  then  soaked  for  from  twelve  to  eighteen 
hours  in  running  water;  this  removes  excessive  acidity; 
they  are  then  placed  in  test-tubes  and  sterilized  by  steam 
on  two  consecutive  days. 

Blood-serum  and  Ascitic  Fluid  with  and  without  the 
Addition  of  Bouillon.  Blood-serum  is  used  in  the  fluid 
state,  semi-solid,  and  firmly  coagulated.  It  is  used 
alone,  with  66  per  cent,  of  bouillon  and  with  25  per 
cent,  of  bouillon  plus  1  per  cent,  of  glucose.  Ascitic, 
pleuritic,  and  hydrocele  fluids  are  also  used  alone,  with 
bouillon,  or  with  nutrient  agar. 

The  Correction  of  the  Reaction  in  Media. 

Formerly  it  was  customary  to  use  litmus-paper  as 
the  indicator  in  neutralizing  media,  adding  soda  solu- 
tion until  the  mixture  turned  the  red  litmus  slightly 
blue,  and  the  blue  litmus  just  a  tinge  less  blue.  This 
is  still  the  best  method  for  those  who  are  only  going 
to  cultivate  the  common  pathogenic  bacteria  for  diag- 
nostic purposes  or  for  the  development  of  toxin.  Most 
parasitic  bacteria  which  grow  at  all  on  artificial  culture 
media  develop  best  in  them  when  they  have  a  slightly 
alkaline  reaction  to  litmus.  If  a  greater  alkalinity  is 
desired  a  certain  number  of  c.c.  of  normal  soda  solu- 
tion can  be  added  for  each  litre;  if  an  acidity  is  desired, 
normal  hydrochloric  acid  solution  is  added. 

Many  bacteriologists  consider  that  litmus  is  not  deli- 
cate enough  to  be  entirely  satisfactory,  especially  when 
experiments  are  to  be  reported  or  exactly  repeated. 
For  these  purposes  phenolphthalein  has  been  generally 
selected.  A  little  experience  will  show  that  different 
indicators  not  only  differ  in  delicacy,  but  that  they 
react  differently  to  different  substances. 


218  BACTERIOLOGY. 

A  litre  of  bouillon  becomes  on  the  addition  of  1  per 
cent,  of  peptone  more  alkaline  to  litmus,  but  decidedly 
more  acid  to  phenolphthalein.  We  have,  therefore, 
especially  with  the  latter  substance,  to  find  by  growing 
the  bacteria  just  what  reaction  we  want,  and  then  test 
the  fluid  with  phenolphthalein  as  the  indicator.  With 
exactly  similar  materials  we  can  exactly  reproduce  at 
any  time  in  the  future  the  same  reaction,  but  with  dif- 
ferent materials  this  would  be  impossible.  A  bou- 
illon which  contains  1  per  cent,  of  peptone  and  reacts 
neutral  to  litmus  is  about  15  points  acid  to  phenolph- 
thalein— that  is,  15  c.c.  of  normal  soda  solution  must 
be  added  per  litre  to  make  the  bouillon  neutral. 

When  phenolphthalein  is  used  we  must  have  accu- 
rately standardized  solutions  of  caustic  soda  and  hydro- 
chloric acid.  The  test  is  carried  out  as  follows  :  To 
10  c.c.  of  the  hot  nutrient  bouillon  add  one  drop  of  a 
1  :  300  solution  in  alcohol  of  phenolphthalein;  into  this 
is  dropped  slowly  a  4  per  cent,  solution  of  caustic  soda 
until  a  faint  rose-tint  appears.  This  indicates  the  be- 
ginning of  an  alkaline  reaction.  To  make  a  litre  neu- 
tral we  would  add  100  times  as  much  of  the  decinormal 
solution  of  caustic  soda  as  was  required  to  make  10  c.c. 
neutral.  As  a  rule,  we  use  1  per  cent,  peptone  bou- 
illon of  such  an  acidity  that  15  c.c.  of  normal  soda 
solution  must  be  added  to  each  litre  to  make  it  neutral. 

The  Sterilization  of  Different  Media. 

Flasks  and  tubes  of  nutrient  broth  and  agar  are 
easily  sterilized  by  placing  them  in  an  Arnold  steam 
sterilizer  (Fig.  20)  for  from  fifteen  minutes  to  one  hour, 
according  to  the  bulk  of  the  fluid,  upon  two  or  three 
consecutive  days.  They  can  also  be  even  more  cer- 


BACTERIOLOGICAL  TECHNIQUE.  219 

tainly  sterilized  by  putting  them  in  an  autoclave  (Fig. 
21)  at  110°  C.  for  from  fifteen  to  thirty  minutes  on 
two  consecutive  days. 

Gelatin  is  sterilized  in  the  same  manner  except,  as 
already  stated,  the  shorter  times  are  used.  Pro- 
longed heating  destroys  the  congealing  properties  of  the 
gelatin. 

Blood-serum  may  be  sterilized  by  fractional  sterili- 
zation and  remain  fluid,  or  may  be  rendered  solid  by 
the  degree  of  heat  used  in  sterilizing. 

For  the  sterilization  of  fluid  serum  it  is  requisite  that 
it  be  exposed  to  a  temperature  of  from  62°  to  66°  C. 
for  one  hour  on  each  of  six  consecutive  days.  The 
best  apparatus  for  obtaining  and  maintaining  this  tem- 
perature (about  65°  C.)  is  a  small  and  well-regulated 
incubator  or  chamber  surrounded  by  a  water  space, 
into  which  the  tubes  and  flasks  containing  serum  are 
to  be  put  each  day  and  in  which  they  are  to  be  left  for 
the  prescribed  time  after  having  been  warmed  to  the 
desired  temperature. 

Serum  may  be  solidified  and  still  remain  translucent 
at  a  temperature  of  76°  C.,  but  when  heated  to  a  higher 
degree  a  more  definite  coagulation  takes  place,  and  the 
medium  becomes  opaque.  Care  must  be  taken  in  coagu- 
lating blood-serum  at  the  higher  temperatures  to  run 
the  temperature  up  slowly  and  not  to  heat  above  90°  C. 
until  the  serum  has  firmly  coagulated;  for  unless  these 
precautions  are  taken  ebullition  is  likely  to  occur,  which 
will  lead  to  the  formation  of  bubbles  and  an  uneven- 
ness  of  the  surface  upon  which  growth  is  to  be  obtained 
and  studied.  Serum  may  be  solidified  at  the  tempera- 
tures mentioned  in  an  incubator,  water-oven,  or  even 
in  an  Arnold  steam  sterilizer,  with  the  top  covered  by 


220  BACTERIOLOGY. 

a  cloth  instead  of  the  usual  lid,  and  when  coagulated 
firmly  (90°  C.)  the  tubes  and  their  contents  may,  on 
the  following  day,  be  sterilized  in  streaming  steam  at 
100°  C.  without  danger  of  the  subsequent  formation 
of  bubbles.  Koch's  serum  coagulator  (Fig.  22)  is, 
however,  the  most  convenient  apparatus. 

Serum  may  be  preserved  by  placing  it  in  flasks 
which,  after  the  addition  of  5  per  cent,  of  chloroform, 
are  sealed.  When  it  is  to  be  used  it  is  filled  into 

FIG.  22. 


Blood-serum  coagulator. 

sterilized  culture  (test)  tubes  and  sterilized  by  exactly 
the  same  methods  as  are  employed  in  sterilizing  fresh 
serum.  The  chloroform,  being  volatile,  tends  to  dis- 
appear at  ordinary  temperatures,  but  is  quickly  and 
surely  driven  off  at  the  temperatures  used  in  steril- 
izing. 

Serum  may  be  efficiently  sterilized,  when  great  care 
is  used,  by  passing  it  through  a  Pasteur  or  Berkefeld 
filter,  under  pressure.  When  so  treated  the  fluid  is 
very  clear  and  light-colored. 

Flasks,  Dishes,  Tubes,  etc.,  Used  for  the  Preservation  of 
Media  and  for  other  Bacteriological  Purposes.  The  nutri- 


BACTERIOLOGICAL  TECHNIQUE. 


221 


ent  media  are  stored  in  large  quantities  in  round  or  flat- 
bottomed  Erlenmeyer  flasks  (Fig.  23).  From  these,  as 
needed,  glass  tubes  (Fig.  24)  are  filled.  Glass  dishes 
with  covers  (Petri  dishes,  Fig.  27)  and  flat  flasks  are 
used  for  growing  bacteria  in  or  upon  thin  layers  of 
media.  When  small  amounts  of  media  are  taken  fre- 
quently from  flasks,  Pasteur's  flasks  (Fig.  25)  are  of 

FIG.  24. 


FIG.  23. 


FIG.  25. 


Erlenmeyer  flask. 


Pasteur  flask. 


Glass  test-tubes. 

great  convenience.  They  consist  of  a  flask  with  a 
ground-glass  neck,  over  which  fits  a  cap.  This  cap 
may  or  may  not  terminate,  as  desired,  in  a  narrow 
tube,  which  is  plugged  with  cotton.  The  cap  keeps 
the  edges  of  the  flask  free  from  bacteria  and  prevents 
the  cotton  from  sticking. 


222  BACTERIOLOGY. 

The  Methods  of  Obtaining  and  Studying  Pure  Cultures  of 
Single  Species  of  Bacteria. 

In  order  to  study  bacteria,  both  in  culture  media 
and  in  the  living  body,  we  must  separate  those  devel- 
oped from  one  organism  from  all  others  and  study 
them  by  themselves  in  pure  cultures.  In  order  to  do 
this  we  have  to  take  the  greatest  precautions  to  insure 
that  the  materials  that  we  make  use  of  for  the  growth  of 
bacteria,  the  flasks  and  tubes  that  hold  these  materials, 
and  the  instruments  with  which  we  transfer  the  bac- 
teria are  sterile.  We  also  carefully  try  to  prevent  any 
bacteria  entering  from  the  air  or  elsewhere. 

The  Cleansing  and  Sterilization  of  Apparatus. 

In  bacteriological  work  sterilization  is  practically 
always  done  by  means  of  dry  and  moist  heat,  for  no 
antiseptic  substances  can  be  allowed  to  remain  in  any 
of  the  media  used  for  the  growth  of  bacteria  or  on  any 
of  the  apparatus  which  would  come  in  contact  with 
them,  as  such  substances  would  inhibit  the  growth  of 
the  bacteria  which  we  desired  to  study. 

The  platinum  wires  and  loops  used  in  transferring 
bacteria  are  sterilized  by  holding  them  for  a  moment 
until  red-hot  in  a  gas  or  alcohol  flame.  They  should 
not  be  used  until  time  enough  has  elapsed  for  them  to 
cool  sufficienly  not  to  injure  the  bacteria  touched  by 
them.  Knives,  instruments,  etc.,  are,  after  thorough 
cleansing,  placed  in  boiling  1  per  cent,  soda  solution 
for  three  to  five  minutes.  Hypodermic  needles  are 
sterilized  by  boiling  in  soda  solution,  or,  when  this  is 
impossible,  they  are  first  frequently  rinsed  with  boil- 
ing or  with  very  hot  water  and  then  filled  with  a  5  per 


BACTERIOLOGICAL   TECHNIQUE. 


223 


cent,  carbolic  acid  solution  for  at  least  thirty  minutes 
and  then  rinsed  again  with  sterile  water.  New  tubes 
and  flasks  sometimes  require  to  be  washed  in  a  2  per 
cent,  solution  of  nitric  acid,  so  as  to  remove  any  free 
alkali  which  may  be  present.  They  are  finally  thor- 
oughly rinsed  in  pure  water.  Old  tubes,  flasks,  and 
other  glassware  are  boiled  for  about  thirty  minutes  in  a 
5  per  cent,  solution  of  washing  soda  and  then  thoroughly 
rinsed  off  with  water  until  perfectly  clean.  If  neces- 


FIG.  26. 


Dry  heat  sterilizer. 


sary,  any  dirt  clinging  to  the  insides  of  the  flasks 
and  tubes  can  be  removed  by  bristle  brushes  or  suit- 
able swabs.  After  the  tubes  and  flasks  have  been 
thoroughly  cleaned  they  are  plugged  loosely  with  ordi- 
nary cotton  batting,  or,  if  that  is  not  at  hand,  the  more 
expensive  absorbent  cotton.  The  tubes  and  flasks  with 
their  cotton  plugs  and  all  other  glassware  are  sterilized 
by  dry  heat  at  150°  C.  for  one  hour  (Fig.  26). 

The  sterile  tubes  and  flasks  are  filled  with  the  media, 
when  small  quantities  are  used,  by  means  of  a  glass 
funnel.  The  main  precaution  to  be  observed  is  not  to 
let  the  media  soil  the  neck  of  the  tubes  and  flasks,  as 


224  BACTERIOLOGY. 

this  would  cause  the  fibres  of  the  cotton  plugs  to  adhere 
to  the  sides  of  the  tubes  when  the  media  dried  and 
make  it  difficult  to  remove  the  plugs  wholly  when  we 
wished  to  inoculate  the  contents  of  the  tubes. 

The  tubes  and  flasks,  plugged  with  sterile  cotton 
and  full  of  media,  are  put  in  the  steam  sterilizer  for 
one  half  hour  on  three  consecutive  days,  or  in  the 
autoclave  for  twenty  minutes  for  two  consecutive  days. 
A  portion  of  the  tubes  containing  nutrient  agar  are 
laid  in  a  slanted  position  before  cooling,  after  the  final 
sterilization,  so  that  a  larger  surface  may  be  obtained. 

Technique  of  Making  Plate  Cultures. 

When  we  make  cultures  from  any  material,  we  are 
very  apt  to  find  that  instead  of  one  variety  of  bacteria 
only  there  are  a  number  present.  If  such  material 
is  placed  in  fluid  media  contained  in  test-tubes,  we  find 
that  the  different  varieties  all  grow  together  and  be- 
come hopelessly  mixed.  When,  on  the  other  hand,  the 
bacteria  are  placed  on  solid  media  they  develop  about  the 
spot  where  they  were  inoculated.  If  different  varieties, 
however,  are  placed  too  near  together,  they  overgrow 
one  another ;  it  is  thus  advisable  to  have  a  greater  sur- 
face of  nutrient  material  than  is  given  on  the  slanted 
surface  of  nutrient  agar  or  blood-serum  contained  in 
test-tubes.  This  need  is  met  by  pouring  the  media 
while  warm  on  flat,  cool,  glass  plates  or  into  shallow 
dishes.  In  making  plate  cultures  two  methods  are 
carried  out.  In  the  first  the  material  with  its  contained 
bacteria  is  scattered  throughout  the  fluid  before  it 
hardens;  in  the  second  it  is  streaked  over  the  surface  of 
the  medium  after  it  has  solidified.  Nutrient  agar  and 
nutrient  gelatin,  the  two  substances  used  for  plate 


BACTERIOLOGICAL  TECHNIQUE.  225 

cultures,  differ  in  two  essential  points,  which  cause 
some  difference  in  their  uses.  Nutrient  1  per  cent, 
agar  melts  at  a  high  temperature  and  begins  to  thicken 
at  about  36°  C.  It  is  not  liquefied  by  bacterial  fer- 
ments. Nutrient  10  per  cent,  gelatin  melts  at  the  low 
temperature  of  about  23°  C.  and  solidifies  at  a  point 
slightly  below  that.  It  is  liquefied  by  many  bacterial 
ferments.  When  we  wish  to  inoculate  fluid  nutrient 
agar  for  plate  cultures  we  have  to  take  great  care  that 
in  cooling  it  to  a  point  which  will  not  injure  the  bac- 
teria, about  41°  C.,  we  do  not  allow  it  to  cool  too  much 
and  thus  solidify  and  prevent  our  pouring  it  into  the 
plates.  To  prevent  this,  when  a  number  of  tubes  are 
to  be  inoculated  they  are  placed  while  still  hot  in  a 
basin  of  water  which  has  been  heated  to  about  45°  C. 
When  the  temperature  of  the  agar  in  one  of  the  tubes, 

FIG.  27. 


Petri  dish. 

as  tested  by  a  thermometer,  has  fallen  to  40°  the  water, 
milk,  feces,  bacterial  culture,  or  other  substances  to  be 
tested  are  added  to  the  other  tubes  in  whatever  quantity 
is  thought  to  be  proper.  After  inoculation  the  con- 
tents of  the  tubes  are  thoroughly  shaken  and  poured 
out  quickly  into  round,  flat-bottomed  glass  dishes  (Fig. 
27),  the  covers  of  which  are  removed  for  the  required 
time  only.  The  bacteria  are  now  scattered  throughout 
the  fluid,  and  as  it  quickly  solidifies  they  are  fixed 
wherever  they  happen  to  be,  and  thus  as  each  individual 

15 


226  BACTERIOLOGY. 

multiplies  clusters  are  formed  about  it  at  the  spot  where 
it  was  fixed  at  the  moment  of  solidification.  The  num- 
ber of  colonies  of  bacteria  (Fig.  28)  thus  indicate  to 
us  roughly  the  number  of  living  bacteria  in  the  quan- 
tity of  fluid  added  to  the  liquid  agar.  Nutrient  gelatin 
is  used  exactly  as  agar,  except  that,  as  it  does  not 
congeal  until  cooled  below  22°  C.,  we  have  no  fear  of 

FIG.  28. 


Photograph  of  a  large  number  of  colonies  developing  in  a  layer  of  gelatin 
contained  in  a  Petri  dish.  Some  colonies  are  only  pin  point  in  size ;  some  as 
large  as  a  pencil.  The  colonies  here  appear  in  their  actual  size. 

its  cooling  too  rapidly.  In  order  not  only  to  count 
the  number  of  colonies  which  develop,  but  also  to 
obtain  a  characteristic  growth,  it  is  desirable  not  to 
have  them  too  near  together.  As  it  is  impossible  to 
determine  accurately  the  number  in  any  suspected  fluid, 
it  is  usual  to  make  a  set  of  four  different  plates,  to  each 
of  which  a  different  amount  of  material  is  added,  so  that 


BACTERIOLOGICAL  TECHNIQUE.  227 

some  one  of  the  four  will  have  the  required  number  of 
colonies.  In  the  first  tube  we  place  an  amount  which  we 
believe  will  surely  contain  sufficient  and  probably  too 
many  bacteria.  To  the  second  tube  we  add  10  per  cent, 
of  the  amount  added  to  the  first,  and  to  the  third  10 
per  cent,  of  the  second,  and  to  the  fourth  10  per  cent, 
of  the  third.  Thus  if  the  first  contained  60,000  colo- 
nies, the  second  would  have  6000  (Fig.  28),  the  third 
600,  and  the  fourth  60.  If,  on  the  other  hand,  the  first 
contained  but  60,  the  second  would  have  about  6,  and 
the  remaining  two  would  probably  contain  none  at  all. 
When  there  are  many  colonies  present  the  dishes  are 
covered  by  a  glass  plate  (Fig.  29),  ruled  in  larger  and 

FIG.  29. 


WolffhiigePs  apparatus  for  counting  colonies. 

smaller  squares.  With  a  hand  lens  the  colonies  in  a 
certain  number  of  squares  are  counted  and  then  the 
number  for  the  whole  contents  estimated. 

When  the  material  to  be  tested  is  crowded  with  bac- 
teria it  is  often  best  to  make  an  emulsion  of  a  portion 
of  it,  and  use  this  rather  than  the  original  substance 
for  making  the  cultures. 

Measured  quantities  of  the  diluted  material  can  be 
transferred  most  accurately  through  a  sterilized  long 
glass  pipette  graduated  in  one-hundredth  cubic  centi- 
metres, or,  more  roughly,  by  a  platinum  loop  of  known 
size. 


228  BACTERIOLOGY. 

The  nutrient  agar-agar  is  frequently  used  in  a  dif- 
ferent manner.  A  small  quantity  is  poured  into  the 
Petri  dish  and  allowed  to  harden.  The  substance  to 
be  tested  bacteriologically,  or  a  dilution  of  it,  is  then 
streaked  by  means  of  a  platinum  loop  lightly  over  its 
surface.  While  in  the  former  method  most  of  the  bac- 
teria developed  under  the  surface,  here  all  develop  upon 
it.  This  is  an  advantage,  as  many  forms  of  bacteria 
develop  more  characteristically  on  the  surface  than  in 
the  midst  of  the  media,  and  it  is  easier  to  remove  them 
free  from  other  bacteria  with  the  platinum  needle.  The 
method  of  using  glass  plates  upon  a  cooling  stage  has 
now  been  practically  given  up  for  the  more  convenient 
one  of  Petri  dishes.  In  warm  weather  the  dishes  should 
be  cooled  before  using,  so  as  to  harden  quickly  the  agar 
or  gelatin  that  is  poured  into  them. 

An  old  method,  which  is  still  sometimes  used  to  find 
the  number  of  living  bacteria,  is,  instead  of  pouring 
out  the  media  which  has  been  inoculated,  to  congeal  it 
on  the  sides  of  the  test-tube.  This  is  best  done  by 
laying  the  tube  flat  on  its  side  on  a  cake  of  ice  and 
rotating  it.  Tubes  come  especially  formed  for  this  by 
having  a  slight  neck,  which  prevents  the  media  run- 
ning up  to  the  plugged  end  of  the  tube.  This  method, 
Esmarch's,  is  used  only  when  the  Petri  dishes  are  not 
obtainable  or  cannot  easily  be  transported. 

The  Study  of  Colonies  in  Plate  Cultures  in  Nutrient  Agar. 

The  plates  should  be  removed  after  twelve  to  twenty- 
four  hours'  growth  at  blood  temperature  and  after  one 
to  three  days  at  70°.  The  special  time  allowed  varies 
according  to  the  rapidity  of  the  growth  of  the  varieties 
developing,  thus  bacteria,  such  as  the  streptococci  and 


BACTERIOLOGICAL  TECHNIQUE.  229 

influenza  bacilli,  reach  the  maximum  development  of 
their  colonies  in  from  ten  to  sixteen  hours,  while  others 
continue  to  spread  for  several  days.  If  we  wait  too  long 
where  numerous  varieties  of  bacteria  are  growing  the 
colonies  of  heavier  growth  may  cover  up  the  finer  and 
more  delicate  ones.  As  a  rule,  the  younger  colonies 
are  more  characteristic,  except  where  the  development 
of  pigment  is  sought. 

FIG.  30. 


Two  surface  colonies  of  diphtheria  bacilli  upon  agar.    X  500  diameters. 

The  colonies  are  first  examined  with  the  eye  (Fig.  28), 
then  with  a  low  magnification,  and  then  again  at  from 
400  to  500  diameters  (Fig.  30).  We  note  everything 
we  can  about  them,  such  as  their  size,  border,  density, 
color,  and  granular  appearance.  At  the  higher  mag- 
nification we  begin  to  detect  the  individual  bacteria. 
After  studying  the  colonies  we  remove  a  few  of  the  bac- 
teria from  one  or  more  of  them  by  touching  them  with 


230  BACTERIOLOGY. 

the  tip  of  a  sterile  platinum  needle,  and  thus  transfer 
them  to  a  cover-glass  for  microscopical  examination,  or 
to  new  media  where  they  may  develop  in  pure  cultures 
and  show  their  growth  characteristics. 

The  Study  of  Plate  Cultures  in  Gelatin  Media. 

The  gelatin  media  have  one  marked  characteristic  to 
be  noted  which  never  occurs  upon  agar — namely,  some 
of  the  colonies  will  be  lying  in  or  surrounded  by  slightly 
opaque  fluid,  due  to  the  liquefaction  of  the  gelatin. 
(See  paler  and  larger  colonies  in  Fig.  28.)  In  using 
nutrient  gelatin  one  must  always  remember  not  to  allow 
it  to  stay  where  the  temperature  is  over  20  C.,  for  if 
that  happens  the  media  will  melt,  nor  must  the  lique- 
fying colonies  be  allowed  to  grow  for  too  long  a  time, 
or  the  entire  media  will  become  fluid. 

FIG.  81. 


Stab  cultures  of  three  cholera  spirilla  in  gelatin,  showing  in  upper  portion 
of  growth  considerable  liquefaction  of  nutrient  gelatin. 

Pure  Cultures.     If  we  transfer  without  contamination 
bacteria  from  a  colony  formed  from  a  single  organism 


BACTERIOLOGICAL  TECHNIQUE.  231 

to  new  media,  and  these  grow,  we  have  what  we  call  a 
pure  culture  of  that  variety.  When  these  are  trans- 
ferred to  the  solid  media  we  call  the  growth  which 
takes  place  from  smearing  the  bacteria  over  the  surface, 
a  surface  or  smear  culture,  and  that  formed  in  the 
depth  of  the  media  by  plunging  the  needle  carrying 
the  bacteria  into  it,  a  stab  culture.  (Fig.  31.) 

In  transferring  bacteria  from  one  tube  to  another  we 
slant  the  tubes  so  that  no  dust  may  fall  within  and 
contaminate  with  other  bacteria  the  special  variety  we 
wish  to  transplant.  The  greatest  care  must  be  taken 
that  the  sterilized  platinum  needle  used  to  transfer  the 
bacteria  is  not  infected  by  touching  any  non-sterile 
matter.  Even  with  our  utmost  care  bacteria  will  from 
time  to  time  pass  from  the  air  or  edges  of  our  tubes 
into  the  culture  media,  and  thus  possibility  of  contami- 
nation must  always  be  kept  in  mind.  When  it  occurs 
upon  solid  media  we,  as  a  rule,  easily  detect  it,  for  we 
notice  the  growth  at  some  point  of  bacteria  of  different 
colony  characteristics;  but  in  fluid  media,  on  account  of 
the  complete  mingling  of  the  bacteria,  we  are  not  so  apt 
to  notice  the  additional  growth. 

Incubators.  In  order  to  have  a  constant  and  proper 
temperature  for  the  growth  of  bacteria,  forms  of  appa- 
ratus called  incubators  have  been  devised.  These  (Fig. 
32)  consist  in  their  simplest  form  of  an  inner  air  cham- 
ber surrounded  by  a  double  copper  wall  containing  water. 
The  apparatus  externally  is  lined  with  asbestos,  to  pre- 
vent radiation.  It  is  supplied  with  doors  and  with 
openings  for  thermometers  and  a  thermo-regulator.  The 
ther mo-regulators  are  of  various  kinds;  those  in  most 
use  depend  upon  the  expansion  or  contraction  of  the 
fluid  in  a  bulb  (A,  Fig.  33),  which  rests  within  the 


232 


BACTERIOLOGY. 


water-jacket,  to  lessen  or  increase  the  space  between 
the  surface  of  the  mercury,  B,  and  the  inner  tube,  D, 
thus  allowing  of  the  passage  of  a  greater  or  less  quan- 
tity of  gas  to  the  burner  through  the  tube  D  (Fig.  33). 


FIG.  33. 


FIG.  32. 


Small  incubator. 


Thermo-regulator. 

Other  forms  depend  upon  the  contraction  or  expansion 
of  metal,  or  the  use  of  the  electric  current  to  control 
the  flow  of  the  2;as. 

O 

The  temperature  in  the  air  chamber  is  kept  above 
that  of  the  surrounding  air  by  means  of  a  gas  flame 
regulated  as  above  described,  or,  when  that  cannot  be 
obtained,  a  lamp. 

The  temperature  is  reduced  by  passing  a  stream  of 
cool  water  through  the  water  chamber,  which  is  itself 
regulated.  When  very  accurate  investigations  are  to  be 


BACTERIOLOGICAL  TECHNIQUE.  233 

made  a  gas-pressure  regulator  is  added  to  the  thermo- 
regulator.  Incubators  are  also  both  warmed  and  regu- 
lated by  electricity. 

In  emergencies  a  culture  may  be  developed  at  the 
blood  temperature  by  placing  it  in  water  contained  in 
a  small  vessel,  which  itself  is  contained  in  a  larger 
vessel,  also  filled.  By  adding  a  little  hot  water  from 
time  to  time  the  temperature  can  readily  be  kept  be- 
tween 34°  and  38°  C.,  which  is  sufficiently  uniform  for 
bacteria  such  as  the  diphtheria  bacilli  to  grow. 

As  a  temporary  expedient  during  the  night,  when 
haste  is  necessary,  it  is  possible, when  the  culture  medium 
is  solid  and  within  a  strong  glass  tube  or  metal  case, 
to  make  use  of  the  body  heat  by  putting  it  under  the 
clothing  next  to  the  body  and  sleeping  upon  it.  Natu- 
rally, this  should  only  be  done  when  other  means  fail. 
Several  times,  when  in  the  country,  this  method  has 
enabled  the  writer  to  obtain  a  growth  of  diphtheria 
bacilli  over  night,  and  thus  get  important  information, 
when  otherwise  it  would  have  been  impossible. 

Culture  Methods  for  Anaerobic  Bacteria. 

Anaerobic  bacteria  will  scarcely  be  cultivated  except 
in  bacteriological  laboratories,  where  the  technique  is 
already  understood,  so  that  the  methods  employed  in 
their  culture  will  only  be  touched  on  here.  A  simple 
device  is  that  of  Koch,  who  placed  a  thin  strip  of 
sterile  mica  upon  the  still  fluid  agar  or  gelatin  in  the 
Petri  dish,  which  had  already  been  inoculated.  After 
the  solidification  of  the  media  the  portion  under  the 
mica  is  excluded  from  the  air  and  anaerobic  growth  can 
develop.  A  second  simple  method  (Liborius)  is  to  fill 
the  tubes  with  media  fuller  than  usual  and  to  inoculate 


234  BACTERIOLOGY. 

the  bacteria  deep  down  to  near  the  bottom  of  the  tubes 
while  the  media  are  still  semi-solid.  An  anaerobic 
growth  will  take  place  in  the  lower  part  of  the  tube. 
In  a  similar  way  the  closed  arm  of  the  fermentation 
tube  will  suffice  for  anaerobic  growth,  if  the  opening 
connecting  it  with  the  open  bulb  is  quite  small.  In 
the  more  complicated  methods  the  plates  or  tubes  are 
placed  in  jars  (Fig.  34),  in  which  the  oxygen  is  dis- 

FIQ.  34. 


Jar  for  anaerobic  cultures. 


placed  by  a  stream  of  hydrogen  developed  by  the  Kipp 
apparatus  through  the  action  of  pure  granulated  zinc 
and  a  pure  25  per  cent,  solution  of  sulphuric  acid. 
When  all  the  oxygen  has  been  displaced  the  jars  are 
sealed  by  rotating  the  stopper.  In  another  method  the 
oxygen  is  extracted  by  a  mixture  of  pyrogallic  acid  and 
caustic  potash.  To  each  100  c.c.  of  air  space  in  the  jar 
1  gramme  of  pyrogallic  acid  and  10  c.c.  of  6  per  cent, 
solution  of  potassium  hydroxide  are  added  and  the  jars 
immediately  sealed.  When  spores  are  present,  a  simple 
method  suggested,  I  believe,  by  McFarland  can  be 
successfully  employed.  Vessels  plugged  with  stoppers 


BACTERIOLOGICAL  TECHNIQUE.  235 

perforated  by  glass  tubes  drawn  to  a  point  are  filled  to 
such  a  height  that  when  the  fluid  is  heated  to  80°  C. 
it  will  just  fill  them.  They  are  inoculated  when  the 
bouillon  is  at  about  60°  C.,  heated  to  80°  C.,  and  then 
sealed  by  closing  the  tube's  point  by  means  of  a  flame. 
After  inoculating  and  heating,  instead  of  sealing  the 
glass  tube  a  sterile  rubber  cork  can  be  inserted.  If 
much  fermentation  is  expected,  the  cork  should  be 
clamped  or  tied  to  the  bottle,  so  that  it  will  not  blow 
out.  One  advantage  of  this  method  is  that  any  con- 
taminating organisms  which  have  no  spores  will  be 
killed.  When  sealed  the  bottles  should  be  cooled  and 
then  placed  in  the  incubator. 


CHAPTER  XIY. 

THE   USE   OF   ANIMALS   FOR   DIAGNOSTIC   AND   TEST 
PURPOSES. 

SUITABLE  animals  are  necessarily  employed  for  many 
bacteriological  purposes.  Thus  they  may  be  used  as  a 
soil  for  bacterial  growth,  when,  as  in  the  case  of  tubercle 
bacilli,  the  bacteria  will  not  develop  in  the  dead  culture 
media.  For  this  reason  material  suspected  to  contain 
tubercle  bacilli  is  injected  into  rabbits  or  guinea-pigs, 
with  the  knowledge  that,  if  present,  although  in  too 
small  numbers  to  be  detected  by  microscopical  or  cul- 
ture methods,  they  will  develop  their  lesions  in  the 
animal's  bodies,  and  thus  reveal  themselves.  The  same 
may  be  true  of  glanders  and  anthrax  bacilli  and  of 
other  bacteria.  Again,  animals  are  used  to  test  the 
virulence  of  organisms,  where,  as  in  the  case  of  diph- 
theria, we  have  very  virulent,  attenuated,  and  non- 
virulent  bacilli  of,  so  far  as  we  know,  identical  cultural 
characteristics.  Here  the  injection  of  a  susceptible 
animal,  such  as  the  guinea-pig,  is  the  only  way  that  we 
can  differentiate  between  those  capable  of  producing 
diseases  from  those  that  are  harmless.  Still  another 
use  of  animals  is  to  differentiate  between  two  virulent 
organisms,  which,  though  entirely  different  in  their 
specific  disease-poisons,  are  yet  so  closely  allied  mor- 
phologically and  in  culture  characteristics  that  they 
cannot  always  be  separated  except  by  studying  their 


THE  INOCULATION  OF  ANIMALS.  237 

action  in  the  animal  body  both  without  and  under  the 
influence  of  specific  serums  upon  them.  In  this  way 
the  typhoid  and  colon  bacilli  may  be  separated,  or  the 
pneumococcus  and  streptococcus.  Still,  a  different  use 
of  animals  is  to  measure  the  protective  effect  of  anti- 
toxic and  bactericidal  serums;  thus,  diphtheria  antitoxin 
is  added  to  diphtheria  toxin  and  injected  into  guinea- 
pigs,  and  streptococcus  immunizing  serum  is  mixed 
with  living  streptococci  and  injected  into  the  vein  of 
a  rabbit.  The  use  of  animals  to  develop  through  bac- 
terial injections  protective  serums  will  be  dealt  with 
under  the  special  bacteria  by  whose  products  they  are 
produced. 

THE  INOCULATION  OF  ANIMALS. 

The   inoculation   of   animals   may   be   made   either 
through  natural  channels  or  through  artificial  ones: 

1.  Cutaneous.     Cultures  are  rubbed  into  the  abraded 
skin. 

2.  Subcutaneous.     The    bacteria    are    injected    by 
means  of  a   hypodermatic  needle   under  the   skin,  or 
are  introduced  by  a  platinum  loop  into  a  pocket  made 
by  an  incision. 

3.  Intravenous.     The  bacteria  are  injected  by  means 
of  a  hypodermatic  needle  into  the  vein.    This  is  usually 
carried  out  in  the  ear  vein  of  the  rabbit.     If  rabbits 
are  placed  in  a  holder,  so  that  the  rabbit  remains  quiet 
and  only  the  head  projects,  it  is  usually  easy  to  pass  a 
small  needle  directly  into  one  of  the  ear  veins,  espe- 
cially those  running  along  their  edges.      If  the  ear  is 
first  moistened  with  a  3  per  cent,  carbolic  acid  solution, 
and  then  supported  between  the  finger  inside  and  the 


238  BACTERIOLOGY. 

thumb  outside,  the  vein  is  usually  clearly  seen  and  en- 
tered with  ease,  if  a  small  sharp  needle  is  held  almost 
parallel  with  the  ear  surface  and  gently  pushed  into  it. 
When  no  holder  is  present,  the  rabbit  can  be  held  by  an 
assistant  seizing  the  forelegs  in  one  hand  and  the  hind 
in  another  and  holding  the  rabbit  head  downward. 

4.  Into  the  anterior  chamber  of  the  eye. 

5.  Into  the  body  cavities.     The  peritoneal  and  less 
often  the  pleural  cavities  are  used  for  bacterial  injec- 
tion.    The  hypodermatic  needle  is  usually  employed, 
less  often  a  glass  tube  drawn  out  to  a  fine  point.     The 
needle  or  the  pointed  glass  tube  are  gently  pushed 
through  the  abdominal  wall,  moved  about  to  insure  its 
freedom  from  the  intestines,  and  the  fluid  injected. 

6.  By  inhalation.     This  method  is  carried  out  by 
forcing  the  animal  to  inhale  an  infected  spray  or  dust. 

7.  By  the  trachea.     This  method  is  carried  out  by 
making  an  incision  in  the  trachea  and  then  inoculating 
the  mucous  membrane  or  injected  substances  into  the 
trachea  and  bronchi. 

8.  Through  the  intestinal  tract  by  swallowing. 

'  In  these  injections  guinea-pigs  are  held,  as  a  rule, 
by  an  assistant  grasping  in  one  hand  the  forelegs  and 
in  the  other  the  hindlegs. 

Rabbits  can  be  held  in  the  same  manner,  or  better 
placed  in  some  holder. 

Mice,  which  are  usually  inoculated  subcutaneously  at 
the  root  of  the  tail,  are  best  placed  in  a  mouse  holder, 
but  can  be  inoculated  by  grasping  the  tail  in  a  pair  of 
forceps,  and  then,  while  allowing  the  mouse  to  hang 
head  downward  in  a  jar,  a  glass  plate  is  pushed  across 
the  top  until  only  space  for  its  tail  is  left. 

All  these  methods  must   be   carried  out  with  the 


THE  INOCULATION  OF  ANIMALS.  239 

greatest  care  as  to  cleanliness,  the  hair  being  clipped 
and  the  skin  partially,  at  least,  disinfected.  After  the 
inoculations  the  animals  should  be  given  the  best  of 
care,  unless,  for  special  purposes,  we  want  to  study 
them  under  unusual  conditions.  For  food,  rabbits 
and  guinea-pigs  require  only  carrots  and  hay. 

If  animals  die,  autopsy  should  be  made  at  the  earliest 
moment  possible,  for  soon  after  death  some  of  the  spe- 
cies of  the  bacteria  in  the  intestines  are  able  to  penetrate 
through  the  intestinal  walls  and  infect  the  body  tissues. 
If  delay  is  unavoidable,  the  animals  should  be  placed 
immediately  in  a  cold  place.  In  making  cultures  from 
the  dead  bodies  the  greatest  care  should  be  taken  to 
avoid  contamination.  The  skin  should  be  disinfected, 
and  any  dust  prevented  by  means  of  a  5  per  cent,  solu- 
tion of  carbolic  acid.  All  instruments  are  sterilized 
by  boiling  in  3  per  cent,  soda  solution  for  five  minutes. 
Changes  of  knives  should  be  made  as  frequently  as  the 
old  ones  become  infected.  When  organs  are  examined 
the  portion  of  the  surface  through  which  an  incision  is 
to  be  made  must  be  sterilized,  if  there  is  danger  that 
the  surrounding  cavity  is  infected,  by  searing  with  the 
flat  blade  of  an  iron  spatula  which  has  been  heated  to 
a  dull  red  heat. 

When  it  is  necessary  to  transport  tissues  some  dis- 
tance they  should  be  wrapped  in  bichloride  cloths  and 
sent  to  the  point  of  destination  as  soon  as  possible.  In 
warm  weather  they  may  be  kept  cool  by  surrounding 
the  vessel  which  contains  them  with  ice. 

Animals  rarely  show  the  same  gross  lesions  as  man 
when  both  suffer  from  the  same  infection.  The  cell 
changes  are  similar,  and,  also,  so  far  as  we  can  test  them, 
the  curative  or  immunizing  effects  of  protective  serums. 


CHAPTER   XV. 

THE  PROCURING  OF  MATERIAL  FOR  BACTERIOLOGICAL 
EXAMINATION  FROM  THOSE  SUFFERING  FROM 
DISEASE. 

A  LONG  experience  has  taught  me  that  physicians 
very  frequently  take  a  large  amount  of  trouble,  and 
yet,  on  account  of  not  carrying  out  certain  simple  but 
necessary  precautions,  make  worthless  cultures  or  send 
material  almost  useless  for  bacteriological  study. 

In  making  cultures  from  diseased  tissues  various  pro- 
cedures may  be  carried  out,  according  to  the  facilities 
which  the  physician  has  and  the  kind  of  information 
that  he  desires  to  obtain.  From  the  dead  body  culture 
material  should  be  removed  at  the  first  moment  possible 
after  death.  Every  hour's  delay  makes  the  results  less 
reliable.  From  both  dead  and  living  tissues  the  less 
the  alteration  that  occurs  in  any  substance  between  its 
removal  from  the  body  and  its  inoculation  upon  or  in 
culture  media  or  animals  the  more  exact  the  informa- 
tion which  will  be  obtained  from  its  examination.  If 
the  material  is  allowed  to  dry  many  bacteria  will  be 
destroyed  in  the  process,  and  certain  forms  which  were 
present  will  be  obliterated,  or,  at  least,  entirely  altered 
in  the  proportion  which  they  bear  to  others.  If  pos- 
sible, therefore,  culture  media  should  be  inoculated 
in  the  neighborhood  of  the  patient  or  dead  body.  For 
that  purpose  a  bacteriologist  should  take  the  most  suit- 


MATERIAL  FOR  EXAMINATION  IN  DISEASE.     241 

able  of  the  culture  media  to  the  bedside  or  autopsy  table. 
Such  a  list  of  media,  if  fairly  complete,  would  comprise 
nutrient  bouillon  alone  and  mixed  with  one-third  its 
quantity  of  ascitic  fluid,  slanted  nutrient  agar,  and 
firmly  solidified  slanted  blood -serum.  If  only  one 
variety  of  media  is  to  be  used  the  solidified  blood- 
serum  is  most  useful  for  parasitic  bacteria,  and  this  can 
be  easily  carried  by  the  physician  and  inoculated  by 
him,  even  if  he  is  not  very  familiar  with  bacterio- 
logical technique.  The  material  must  be  obtained  in 
different  ways,  according  to  the  nature  of  the  infection. 
For  the  detection  of  the  bacteria  causing  septicsernia 
we  are  met  with  the  difficulty  that  there  are  apt  to  be 
very  few  or  no  organisms  present  in  the  blood  until 
shortly  before  death.  It  will,  therefore,  be  useless  to 
take  only  a  drop  of  blood  for  cultures,  as  even  when 
present  there  may  not  be  more  than  eight  or  ten  organ- 
isms in  a  cubic  centimetre.  If  cultures  are  to  be  made 
at  all,  it  is,  therefore,  best  to  make  them  correctly  by 
taking  from  3  to  5  c.c.  of  blood  by  means  of  a  sterile 
hypodermatic  needle,  or  a  suitable  glass  tube  armed 
with  a  hypodermatic  needle,  from  the  vein  of  the  arm, 
after  proper  cleansing  of  the  skin  and  a  tiny  incision. 
Into  each  of  five  different  tubes  containing  bouillon  we 
add  one-fifth  of  the  quantity  of  blood  withdrawn.  We 
have  made  by  this  mixture  of  blood  and  bouillon  a  most 
suitable  medium  for  the  growth  of  all  bacteria  which 
produce  septicaemia,  and  at  the  same  time  have  added  a 
sufficient  quantity  of  blood  to  insure  us  the  best  possible 
chance  of  having  added  some  of  the  bacteria  producing 
the  disease.  We  also  streak  several  nutrient  agar  plates 
with  blood,  so  as  to  indicate  roughly  the  number  of 
organisms  present,  if  they  happen  to  be  in  abundance, 

16 


242  BACTERIOLOGY. 

From  wounds,  abscesses,  cellulitis,  etc.,  the  substance 
for  bacteriological  examination  can,  as, a  rule,  best  be 
obtained  by  means  of  small  rods  armed  with  a  little 
absorbent  cotton.  A  number  of  these  can  be  carried 
in  a  test-tube.  Both  rods  and  tubes  must  be  sterile. 
The  swab  is  inserted  in  the  wound,  then  streaked 
gently  over  the  oblique  surface  of  the  nutrient  agar  in 
one  tube,  over  the  blood-serum  in  another,  and  then 
inserted  in  the  bouillon.  Finally,  either  at  the  bed- 
side or  in  the  laboratory,  material  is  thinly  streaked 
over  the  surface  of  nutrient  agar  contained  in  several 
Petri  dishes.  We  inoculate  several  varieties  of  media, 
with  the  hope  that  one  at  least  will  prove  a  suitable 
soil  for  the  growth  of  the  organisms  present.  From 
surface  infections  of  mucous  membranes,  as  in  the 
nose,  throat,  vagina,  etc.,  the  swab,  again,  is  probably 
the  most  useful  instrument  for  obtaining  the  mate- 
rial for  examination.  The  greatest  care,  of  course, 
must  be  used  in  all  cases  to  remove  the  material  for 
study  without  contaminating  it  in  any  way  by  other 
material  which  does  not  belong  to  it.  Thus,  for  in- 
stance, if  we  wish  to  obtain  material  from  an  abscess 
of  the  liver,  where  the  organ  lies  in  a  peritoneal  cavity 
iufected  with  bacteria,  here  one  must  first  absolutely 
sterilize  the  surface  of  the  liver  by  pressing  on  it  the 
blade  of  a  hot  iron  spatula  before  cutting  into  the  ab- 
scess, so  that  we  may  not  attribute  the  infection  which 
caused  the  abscess  to  the  germs  which  we  obtained  from 
the  infected  surface  of  the  liver.  From  such  an  organ 
as  the  uterus  it  is  only  with  the  greatest  care  that  we 
can  avoid  outside  contamination,  and  only  an  expert 
bacteriologist  familiar  with  such  material  will  be  able 
to  eliminate  the  vaginal  from  the  uterine  bacteria. 


MATERIAL  FOR  EXAMINA TION  IN  DISEASE.     243 

A  statement  of  the  conditions  under  which  materials 
are  obtained  should  always  accompany  them  when  sent 
to  the  laboratory  for  examination,  even  if  the  exami- 
nation is  to  be  made  by  the  one  who  made  the  cul- 
tures. These  facts  should  be  noted,  or  otherwise  at 
some  future  date  they  may  be  forgotten  and  mislead- 
ing information  sent  out.  The  work  of  obtaining 
material  for  examination  without  contamination  is  at 
times  one  of  extreme  difficulty.  It  simply  must  be 
remembered  that  if  contamination  does  take  place  our 
results  may  become  entirely  vitiated,  and  if  the  diffi- 
culties are  so  great  that  we  cannot  avoid  it,  it  may 
simply  mean  that  under  such  conditions  no  suitable 
examination  can  be  made.  Where  the  substance  to 
be  studied  cannot  be  immediately  subjected  to  cultures 
or  animal  inoculations,  it  should  be  transferred  in  a 
sterile  bottle  as  soon  as  possible  to  a  location  where  the 
cultures  can  be  made.  If  for  any  reason  delay  must 
take  place,  the  material  should  at  least  be  put  in  a 
refrigerator,  where  cold  will  both  prevent  any  further 
growth  of  some  varieties  of  bacteria  and  lessen  the 
danger  of  the  death  of  others.  After  having  made 
the  cultures,  some  of  the  infected  material  should 
always  be  smeared  on  a  couple  of  clean  slides  or  cover- 
glasses  and  allowed  to  dry.  These  can  be  stained  and 
examined  later,  and  may  give  much  valuable  informa- 
tion. 

In  obtaining  samples  of  fluid,  such  as  urine,  feces, 
etc.,  the  bottles  in  which  they  are  placed  should  always 
be  sterile,  and,  of  course,  no  antiseptic  should  be  added. 
It  is  necessary  to  clearly  explain  this  to  the  nurse,  for 
she  has  probably  been  instructed  to  add  disinfectants 
to  all  discharges.  Disinfected  material  is,  of  course, 


244  BACTERIOLOGY. 

entirely  useless  for  bacteriological  investigations.  It 
cannot  be  too  much  emphasized  that  materials  which 
are  not  immediately  used  should  be  sent  to  the  labora- 
tory as  quickly  as  possible,  for  in  such  substances  as 
feces,  where  enormous  numbers  of  various  kinds  of 
bacteria  are  present,  those  which  we  seek  most,  such  as 
the  typhoid  bacilli,  frequently  succumb  to  the  delete- 
rious products  of  the  other  bacteria  present.  Even 
when  abundantly  present  living  typhoid  bacilli  may 
entirely  disappear  from  the  feces  in  the  course  of  even 
twelve  hours,  while  at  other  times  they  may  remain 
present  for  weeks.  These  differences  depend  on  the 
associated  organisms  present,  the  chemical  constitution 
of  the  feces  or  urine,  and  the  conditions  under  which 
the  material  is  obtained. 


CHAPTER   XVI. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER  AND 
AIR — THE  CONTAMINATION  AND  PURIFICATION 
OF  DRINKING  WATERS. 

Bacteriological  Examination  of  Water.  The  bacterio- 
logical examination  of  water  is  undertaken  with  two 
purposes  :  First,  to  discover  the  number  of  living  bac- 
teria present  in  the  water,  and,  second,  the  varieties 
that  may  be  present.  In  order  to  roughly  determine 
the  number  of  living  bacteria  in  water,  we  thoroughly 
mingle  certain  definitely  measured  quantities  of  water 
with  suitable  quantities  of  melted  but  sufficiently  cooled 
nutrient  agar  or  gelatin,  the  mixtures  being  immedi- 
ately poured  into  Petri  dishes,  or  retained  in  Esmarch 
tubes  and  allowed  to  quickly  harden.  The  bacteria  in 
the  water  are  thus  scattered  throughout  the  solidified 
media.  If  nutrient  gelatin  is  employed  care  must  be 
taken  to  keep  it  cool  during  transportation.  The  agar 
is  usually  allowed  to  remain  at  the  body-temperature, 
while  the  gelatin  is  necessarily  kept  at  the  usual  room- 
temperature  (about  70°  F.).  If  nutrient  agar  alone 
is  used  two  sets  of  plates  are  made,  one  being  kept  at 
body-heat  the  other  at  the  usual  room-temperature. 
After  a  suitable  length  of  time  has  been  allowed  for 
the  development  of  the  colonies  we  examine  the  plate 
cultures,  and  by  counting  the  number  of  colonies  de- 
veloped by  means  of  a  low  power  lens  and  Wolffhiigel's 
apparatus  (Fig.  29)  or  some  equivalent,  we  are  enabled 
to  find  approximately  the  number  of  living  bacteria 


246  BACTERIOLOGY. 

which  were  present  in  the  water  at  the  moment  the 
water  was  examined.  Any  bacteria,  however,  which, 
though  living  in  the  water,  were  unable  to  grow  in  the 
media  or  at  the  temperatures  employed  would,  naturally, 
not  reveal  themselves  by  the  growth  of  colonies,  nor 
would  bacteria  clinging  together  in  bunches  count  as 
more  than  a  single  member.  As  to  the  value  of  learn- 
ing the  number  of  bacteria  in  water,  we  must  admit  that 
a  single  determination  of  the  number  of  living  bacteria 
in  any  sample  is  now  known  to  be  of  little  avail  unless 
the  conditions  under  which  the  water  exists  are  well 
known  or  the  number  of  bacteria  is  enormous.  Thus, 
for  instance,  the  water  in  an  Adirondack  lake  might 
contain  in  a  cubic  centimetre  far  more  bacteria  than 
that  of  a  well  which  was  slightly  contaminated  with 
typhoid  bacilli  from  human  sources.  If,  however,  we 
knew  the  usual  condition  of  the  well  water  and  the 
usual  number  of  bacteria  present  in  it,  any  sudden 
increase  would,  of  course,  give  us  a  strong  suspicion, 
but  nothing  more  than  a  suspicion,  of  dangerous  con- 
tamination. In  the  same  way  in  a  stream  into  which 
a  sewer  empties,  if  we  find  a  great  many  more  bacteria 
in  the  stream  some  distance  below  the  point  of  entrance 
of  the  sewer  than  there  were  above,  we  would  have 
every  reason  to  believe  that  the  increase  of  bacteria 
found  in  the  stream  below  was  due  to  the  bacteria  added 
to  the  stream  by  the  sewer;  if  we  drank  that  water  we 
would  know  from  the  examination  we  were  drinking 
not  only  a  portion  of  the  sewage  but  of  the  bacteria 
contained  therein.  It  is  true,  that  with  our  present 
knowledge, derived  from  previous  bacteriological  studies, 
we  would  be  almost  as  certain  of  these  facts  before  as 
after  the  bacteriological  examination.  The  determina- 


EXAMINATION  OF  WATER  AND  AIR.        247 

tion,  therefore,  of  the  number  of  bacteria  should  only 
be  considered  of  value,  except  in  the  extreme  instances 
where  enormous  numbers  are  found,  when  we  know 
fairly  well  the  conditions,  chemical  and  physical,  con- 
cerning the  supply.  The  examination  of  water,  to  de- 
termine whether  or  not  any  forms  of  parasitic  bacteria 
or  other  micro-organisms  are  present,  would  be  more 
often  of  practical  value  than  it  is  if  the  difficulties 
were  not  so  great.  As  a  matter  of  fact,  water  exami- 
nations for  this  purpose  are  usually  negative.  The 
varieties  of  bacteria  most  sought,  except  in  the  presence 
of  a  cholera  epidemic,  are  the  typhoid  and  colon  bacilli. 
If  it  were  possible  to  readily  obtain  the  typhoid  bacilli 
from  water,  when  they  were  present  in  small  numbers, 
its  examination  for  that  purpose  would  be  of  much 
greater  value  than  it  is  now;  but  we  have  to  remem- 
ber that  we  can  only  examine  at  one  time  a  few  cubic 
centimetres  of  water  by  bacteriological  methods,  and 
that  although  the  typhoid  bacilli  may  be  sufficiently 
abundant  in  the  water  to  give,  in  the  quantity  that  we 
ordinarily  drink,  a  few  bacilli,  yet  it  must  be  a  very 
lucky  chance  if  they  happen  to  be  in  the  small  amount 
which  we  examine.  Still,  further,  although  it  is  very 
easy  to  isolate  typhoid  bacilli  from  water  when  they 
are  in  considerable  numbers,  yet  when  they  are  a 
very  minute  proportion  of  all  the  bacteria  present  it 
is  almost  impossible  not  to  overlook  them.  Many 
attempts  have  been  made  to  devise  some  method  by 
which  the  relative  number  of  the  typhoid  and  other 
parasitic  bacteria  present  in  water  could  be  increased 
at  the  expense  of  the  saprophytic  bacteria.  Thus  to 
100  c.c.  of  water  25  c.c.  of  a  4  per  cent,  peptone 
nutrient  bouillon  is  added,  and  the  whole  put  in  the 


248  BACTERIOLOGY. 

incubator  at  37°  C.  for  twenty-four  hours.  From 
this  plate  cultures  are  made.  In  our  experience  this 
and  other  methods  have  not  enabled  us  to  detect  the 
typhoid  bacillus  where  we  have  failed  to  find  it  by 
making  direct  plate  cultures.  As  a  matter  of  fact,  the 
typhoid  bacillus  is  found  in  such  a  small  number  of  the 
specimens  where  we  actually  know  that  it  is  or  has 
been  present  in  the  water  from  which  they  were  ob- 
tained, because  of  cases  of  typhoid  fever  which  have 
developed  from  drinking  the  water,  that  we  must  con- 
sider our  lack  of  finding  the  bacillus  in  any  given 
case  as  absolutely  no  reason  for  considering  the  water 
to  be  free  from  danger.  Another  serious  drawback 
to  the  value  of  the  examinations  is  that  they  are  fre- 
quently made  at  a  time  when  the  water  is  really  free 
from  contamination,  though  both  earlier  and  later  the 
bacillus  was  present;  it  is  hardly  worth  while,  there- 
fore, except  in  careful  experimental  researches,  to  ex- 
amine the  water  for  the  typhoid  bacillus,  but  rather 
study  the  location  of  the  surrounding  privies  and  sources 
of  contamination.  The  colon  bacilli  are  far  more  easy 
to  detect,  because  they  are  apt  to  be  more  abundant, 
and,  also,  because  they  grow  more  readily  in  artificial 
culture  media.  A  method  suggested  by  Theobald 
Smith  is  of  value  in  both  finding  and  excluding  the 
presence  of  bacilli  of  the  colon  group.  He  adds  a  few 
drops  of  the  suspected  water  to  glucose  nutrient  bouillon 
in  fermentation  tubes,  and  keeps  it  at  37°  C.  for  from 
thirty-six  to  forty-eight  hours.  If  no  fermentation 
occurs  no  colon  bacilli  are  present.  If  it  does  occur 
plates  are  made  and  the  bacteria  isolated  and  tested. 
In  the  bouillon  the  colon  bacilli  when  present  usually 
increase  in  numbers  and  are  then  readily  detected.  The 


EXAMINATION  OF  WATER  AND  A  IE.        249 

presence  of  the  colon  bacilli  in  water  is,  except  possibly 
in  rare  cases,  only  of  importance  as  an  indication  of  fecal 
contamination,  and,  therefore,  of  the  possibility  of  dan- 
gerous infection  with  other  bacteria.  Formerly  it  was 
considered  that  its  presence  indicated  human  fecal  con- 
tamination, but  we  now  know  that  many  of  the  animals 
contain  in  their  intestines  colon  bacilli  so  similar  to  those 
found  in  human  beings  that  we  cannot  differentiate 
the  one  variety  from  the  other;  therefore,  the  finding  of 
colon  bacilli  in  water  must  always  be  judged  according 
to  the  conditions  surrounding  the  water-supply.  Thus, 
it  may  indicate  cattle  contamination  from  the  barn  or 
surface  water,  human  contamination,  or,  in  certain 
conditions,  simply  the  accidental  contamination  of  the 
stream  by  wandering  cattle  or  animals.  Properly 
judged,  however,  the  examination  for  the  colon  bacilli 
may  yield  results  of  considerable  practical  impor- 
tance. Whenever,  in  water  examinations,  any  special 
variety  of  bacteria  is  found  in  unusual  abundance,  the 
fact  should  be  noted, for  sometimes  it  maybe  the  cause 
of  some  prevailing  infectious  disease  ;  thus  the  bacillus 
pyocyaneus  has  been  found  in  water  producing  diar- 
rhoea with  greenish  discharges. 

The  Obtaining  of  Water  for  Examination.  Whenever 
possible  the  inoculation  of  the  gelatin  or  agar  tubes 
and  the  pouring  of  their  contents  into  the  Petri  dishes 
should  be  done  immediately  after  gathering  the  samples, 
otherwise  the  actual  and  relative  numbers  of  the  differ- 
ent organisms  will  change.  As  a  rule,  the  pathogenic 
bacteria  will  decrease  and  the  harmless  water  species 
will  increase.  When  the  plates  cannot  be  made  imme- 
diately the  water  held  in  the  sterile  vials  should  be 
kept  nearly  at  the  freezing-point.  Even  at  very  low 


250  BACTERIOLOGY. 

temperatures  some  forms  of  bacteria  increase  rapidly. 
Unless  the  nutrient  gelatin  or  agar  inoculations  are 
made  within  an  hour  or  two  the  count  of  the  number 
of  colonies  is  practically  useless.  Considerable  care 
is  necessary  in  taking  the  samples  of  water,  so  as  not 
to  get  extraneous  organisms  in  the  water  from  sur- 
rounding sources.  Three  slightly  different  methods 
will  suffice  to  indicate  how  it  should  be  done.  A 
simple  and  accurate  method  of  collecting  the  water  is 
to  have  several  graduated  sterile  glass  pipettes  plugged 

FIG.  35.  FIG.  36.  FIG.  37. 


Bulb  pipette.  Graduated  pipette.  Sternberg  bulb. 

at  the  bottom  by  a  cork  and  above  by  cotton.  This  is 
inserted  the  required  depth,  to  avoid  the  surface  water 
with  its  particles  of  dirt,  and  the  cork  pushed  off  by 
a  second  pipette  or  rod  and  the  water  allowed  to  flow 
or  be  sucked  in.  The  upper  end  is  now  stopped  by 
the  finger  and  the  pipette  removed  and  a  definite 
amount  of  water  tested  (Figs.  35  and  36).  A  simple 
glass  tube,  sterilized  by  passing  it  through  a  flame  and 
corked  below,  will  answer  the  same  purpose,  or,  again, 
a  tube,  one  end  of  which,  after  sealing,  is  blown  into  a 
sphere  and  the  other  end  drawn  out  into  a  capillary  stem 


EXAMINATION  OF  WATER  AND  AIR.        251 

(Fig.  37).  The  stem  must  be  sealed  while  the  bulb  is 
still  hot,  or  while  a  little  water  is  being  boiled  in  it,  so 
that  a  partial  vacuum  may  exist  in  the  bulb,  in  order 
that  the  water  may  be  sucked  up  into  it  when  the 
stem  is  broken.  The  inoculation  of  the  media  is  now 
made  directly,  or  water  from  the  tube  is  emptied  into  a 
sterile  bottle  or  test-tube,  or  the  end  of  the  Sternberg 
bulb  is  sealed  by  heat.  When  water  is  to  be  obtained 
from  greater  depths  or  from  beneath  the  surface  of 
wells,  more  complicated  forms  of  apparatus  are  neces- 
sary. A  good  example  is  the  one  devised  by  Abbott, 

FIG.  38. 


Flask  for  counting  colonies  of  bacteria. 

and  made  for  him  by  Charles  Leotz  &  Sons,  of  Phila- 
delphia. It  consists  of  a  metal  framework  in  which  is 
encased  a  bottle  provided  with  a  ground-glass  stopper. 
To  the  stopper  a  spring  clamp  is  attached,  and  this  in 
turn  is  operated  by  a  string,  so  that  when  the  weighted 
apparatus  is  allowed  to  sink  into  the  stream  the  stopper 
may  be  removed  at  any  depth  desired  by  simply  pulling 
on  the  string.  When  the  bottle  is  full  the  stopper  is 
allowed  to  spring  back  into  position  by  releasing  the 
spring.  Before  removing  the  water  the  neck  of  the 
bottle  should  be  sterilized  by  pouring  a  little  of  a  5 


252  BACTERIOLOGY. 

per  cent,  solution  of  carbolic  acid  upon  it  and  drying 
with  a  sterile  cloth. 

The  technique  of  making  plate  cultures,  of  counting 
the  number  of  colonies,  and  of  isolating  and  identi- 
fying pathogenic  species  are  described  under  the  special 
chapters  devoted  to  these  subjects.  A  point  to  be  re- 
membered is  that  about  double  the  number  of  colonies 
usually  develop  at  20°  C.  as  at  blood  heat  (37°  C.), 
many  water  bacteria  not  growing  at  body-temperature. 
A  convenient  flat  flask  with  ruled  surface  (Fig.  38) 
has  been  devised  to  take  the  place  of  the  Petri  dish 
when  the  number  of  bacteria  only  and  not  the  varieties 
are  wanted.  In  these  there  is  no  danger  of  contami- 
nating air-organisms  entering  during  transportation. 
The  stopper  can  be  graduated  to  hold  one  c.c. 

The  Bacteriological  Examination  of  Air.  Saprophytic 
bacteria  are  always  present  in  considerable  numbers  in 
the  air  except  far  out  at  sea  or  on  high  mountains. 
They  are  more  abundant  where  organic  matter  abounds 
and  in  dry  and  windy  weather.  Pathogenic  bacteria, 
on  the  other  hand,  are  only  occasionally  present  in 
the  air.  The  practical  results  obtained  from  the  ex- 
amination of  air  for  pathogenic  bacteria  have  been 
slight.  We  know  that  at  times  they  must  be  in  the 
air,  but  unless  we  purposely  increase  their  numbers 
they  are  so  few  in  the  comparatively  small  amount  of 
air  which  it  is  practicable  to  examine  that  we  rarely  find 
them.  Examination  of  dust,  however,  in  hospital 
wards  and  sick-rooms,  in  places  where  only  air  infec- 
tion was  possible,  have  revealed  tubercle  bacilli  and 
other  pathogenic  bacteria. 

The  simplest  method  of  searching  for  the  varieties 
of  bacteria  in  the  air  and  their  number  in  any  place  is 


PURIFICATION  OF  WATER.  253 

to  expose  to  the  air  for  longer  or  shorter  periods  nutrient 
agar  spread  upon  the  surface  of  the  Petri  dish.  After 
exposure  the  plates  are  put  either  in  the  incubator  at 
37°  C.  or  kept  at  room-temperature.  The  more  careful 
examination  is  made  by  drawing  a  given  quantity  of 
air  through  tubes  containing  sterile  sand,  which  is  kept 
in  by  pieces  of  metal  gauze.  When  the  operation  is 
completed  the  sand  is  poured  into  a  tube  containing 
melted  nutrient  gelatin  or  nutrient  agar,  and  after 
thoroughly  shaking  the  mixture  is  poured  into  a  Petri 
dish  and  the  bacteria  allowed  to  develop,  either  at 
37°  or  20°  C.,  according  as  a  growth  of  the  parasitic 
or  saprophytic  varieties  is  desired. 

THE    CONTAMINATION   AND    PURIFICATION    OF 
DRINKING   WATERS. 

Brook  and  river  water  is  contaminated  in  two  ways: 
through  chemicals,  the  waste  products  of  manufactur- 
ing establishments,  and  through  harmful  bacteria  by 
the  contents  of  drains,  sewers,  etc.,  the  latter  method 
being  by  far  the  more  dangerous. 

When  water,  which  has  been  soiled  by  waste  products 
of  manufactories  only,  becomes  so  diluted  or  purified 
that  the  contamination  is  not  noticeable  to  the  senses 
and  shows  no  dangerous  products  on  chemical  analysis 
it  is  probably  safe  to  drink.  When  sewage  is  the  con- 
tamination this  rule  no  longer  holds,  and  there  may  be 
no  chemical  impurities  and  no  pathogenic  bacteria 
found  and  yet  disease  be  produced.  That  river  water 
which  has  been  fouled  by  sewage  will,  in  the  course  of  a 
few  miles,  through  the  dilution  of  additional  supplies, 
through  sedimentation,  and  through  oxidation,  become 


254  BACTERIOLOGY. 

greatly  purified  is  an  indisputable  fact.  The  increase  in 
bacteria  which  occurs  from  contamination  is  also  largely 
or  entirely  lost  after  ten  to  twenty  miles  of  river  flow. 
Nevertheless,  the  history  of  many  epidemics  seems  to 
show  that  a  badly  contaminated  river  is  never  a  safe 
water  to  drink,  although  with  the  lapse  of  time  it  be- 
comes less  and  less  dangerous,  nor  will  sand  filter-beds 
absolutely  remove  all  danger.  These  statements  are 
founded  upon  the  results  of  numerous  investigations; 
thus  the  marked  disappearance  of  bacteria  is  illustrated 
by  the  following  :  Kummel  found  below  the  town  of 
Rosbock  48,000  bacteria  to  the  cubic  centimetre ; 
twenty-five  kilometres  further  down  the  stream  only 
200  were  present — about  the  same  number  as  before 
the  sewage  of  Rosbock  entered.  On  the  other  hand, 
the  doubtful  security  of  depending  on  a  river  purifi- 
cation is  proved  by  such  experiences  as  the  following: 
In  the  city  of  Lowell,  Massachusetts,  an  alarming 
epidemic  followed  the  pollution  of  the  Merrimac  River 
three  miles  above  by  typhoid  feces,  and  six  weeks  later 
an  alarming  epidemic  attacked  Lawrence,  nine  miles 
below  Lowell.  It  was  estimated  that  the  water  took 
ten  days  to  pass  from  Lowell  to  Lawrence  and  through 
the  reservoirs.  As  typhoid  bacilli  may  live  for  twenty- 
five  days  in  water,  the  Lawrence  epidemic  is  easily 
explained.  Newark-on-Treut,  England,  averaged 
seventy-five  cases  a  year  from  filtered  water  and  only 
ten  when  it  was  changed  to  deep-well  supply. 

The  Purification  of  Water  on  a  Large  Scale.  Surface 
waters,  if  collected  and  held  in  sufficiently  large  lakes 
or  reservoirs,  usually  become  so  clarified  by  sedimen- 
tation as  to  require  no  further  treatment  so  far  as  its 
appearance  goes.  The  collection  of  water  in  large 


PURIFICATION  OF  WATER.  255 

reservoirs  allows  not  only  the  living  and  dead  matter 
to  subside,  but  allows  time  also  for  the  pathogenic  germs 
to  perish  through  light  and  antagonistic  bacteria  and 
other  deleterious  influences.  Filtration  of  water  ex- 
erts a  very  marked  purification,  taking  out  99  per  cent, 
of  the  organisms  in  those  best  constructed  and  at  least 
90  per  cent,  in  those  commonly  used  in  cities.  The 
construction  of  filters  is  too  large  a  subject  to  enter  on 
minutely  here;  they  consist,  as  a  rule,  of  several  layers, 
beginning  with  fine  sand,  and  then  smaller  and  larger 
gravel,  and  finally  rough  stones.  A  certain  time  elapses 
before  the  best  results  are  obtained;  this  seems  to  wait 
for  the  formation  of  a  film  of  organic  material  on  the 
sand,  which  is  full  of  nitrifying  bacteria.  Even  the 
best  filters  only  greatly  diminish  the  dangers  of  pol- 
luted water.  Spring  and  well  waters  are,  in  fact, 
filtered  waters. 

Domestic  Purification.  Water  which  requires  private 
filtering  should  not  be  supplied  for  drinking  purposes. 
Unhappily, however, it  often  is.  Filters  may  be  divided, 
roughly,  into  those  for  low  and  high  pressure.  The 
former  are  directly  connected  with  the  water  main,  while 
the  others  simply  have  the  slight  pressure  of  the  column 
of  water  standing  in  the  filter.  Many  high-pressure 
filters  contain  animal  charcoal,  silicated  carbon,  etc., 
either  in  a  pressed  condition  or  in  one  porous  mass. 
These  filters  remove  much  of  the  deleterious  matter 
from  the  suspected  waters,  but  the  majority  cannot  be 
depended  upon  to  remove  all  bacteria.  Even  those 
which  are  equipped  for  self -clean  sing  become  in  a  little 
while  foul,  and,  if  not  cleaned,  unfit  for  use.  The  best 
of  the  class  are  the  Berkefeld  and  Pasteur  filters. 
These  yield  a  water,  if  too  great  pressure  is  not  used, 


256  BACTERIOLOGY. 

almost  absolutely  free  from  bacteria,  and  if  they  are 
frequently  cleansed  they  are  reliable.  A  large  Berke- 
feld  filter  will  allow  sixty  gallons  of  water  to  pass  per 
hour.  The  Pasteur  filter  is  more  compact  and  slower. 
From  the  best  Pasteur  filters  sterile  water  may  be 
passed  for  two  to  three  weeks;  from  the  Berkefeld 
usually  only  a  few  days.  A  simple  typical  low- 
pressure  filter  is  that  of  Bailey  Denton.  The  upper 
compartment  contains  the  filtering  material,  which 
may  be  sand  or  charcoal,  and  is  fed  from  a  cistern  or 
hydrant.  After  a  certain  quantity  of  water  has  passed 
in  the  supply  is  automatically  cut  off  until  the  whole 
amount  has  filtered.  A  filter  easily  made  is  the  follow- 
ing :  Take  a  large-sized  earthenware  pot  and  plug  the 
hole  in  the  bottom  with  a  cork,  through  which  pass  a 
short  glass  tube.  Upon  the  bottom  place  an  inch  of 
small  pieces  of  broken  flower-pot;  upon  this  a  couple 
of  inches  of  well-washed  small  gravel,  and  upon  this 
six  to  twelve  inches  of  well- washed  fine,  sharp  sand. 
Cover  the  sand  with  a  piece  of  filter-paper  and  hold 
this  down  with  a  few  small  stones.  Mount  the  pot  on 
a  tripod,  and  it  is  ready  for  use.  The  paper  prevents 
the  sand  being  disturbed  when  water  is  added,  and  as 
it  also  holds  most  of  the  sediment,  this  can  be  readily 
removed.  Every  few  months  the  sand  can  be  washed 
and  replaced.  Animal  charcoal  is  not  a  good  substance 
for  permanent  filters,  as  bacteria  grow  well  in  it. 
Whenever  water  is  suspected,  and  there  is  any  doubt 
as  to  the  filters,  it  should  be  boiled  for  ten  minutes; 
this  will  destroy  all  bacteria.  This  precaution  should 
always  be  taken  in  the  presence  of  typhoid  fever  and 
cholera  epidemics, 


CHAPTER  XVII. 

THE   CLASSIFICATION    OF   BACTERIA. 

THE  PERMANENCE  OF  VARIETIES. 

BACTERIA  have  been  classified  in  many  different 
ways  by  many  different  observers.  As  a  rule,  the  genera 
are  based  upon  morphological  characters  and  the  species 
upon  biochemical,  physiological,  or  pathogenic  prop- 
erties. While  the  form,  size,  and  method  of  division 
are  the  most  permanent  characteristics  of  bacteria,  and 
so  are  naturally  utilized  for  classification,  nevertheless, 
in  this  basis  of  division  there  are  decided  difficulties. 
Thus  while  the  form  and  size  of  bacteria  are  fairly  con- 
stant under  the  same  conditions,  they  are  in  many  quite 
different  under  diverse  conditions.  Another  serious 
drawback  is  that  these  morphological  characteristics 
give  no  indication  whatever  of  the  relations  of  the  bac- 
teria to  disease  and  fermentation — the  very  characteris- 
tics for  which  as  physicians  we  study  them.  Other 
properties  of  bacteria  which  are  fairly  constant  under 
uniform  conditions  are  those  of  spore  and  capsule  for- 
mation, motility,  reaction  to  staining  reagents,  relation 
to  temperature,  to  oxygen  and  other  food  material,  and, 
finally,  their  relation  to  fermentation  and  disease. 

Taking  any  one  of  these  properties  of  bacteria  as  a 
basis,  we  can  classify  them;  but  even  here  there  will 
be  groups  which  under  certain  conditions  would  be 

17 


258  BACTERIOLOGY. 

placed  in  one  class  and  under  others  in  another.  Thus 
the  power  to  produce  spores  may  be  totally  lost  or  held 
in  abeyance  for  a  time. 

The  relations  to  oxygen  may  be  gradually  altered, 
so  that  an  anaerobic  species  grows  in  the  presence  of 
oxygen.  Parasitic  bacteria  may  be  so  cultivated  as  to 
become  saprophytic  varieties,  and  those  which  have 
no  power  to  grow  in  the  living  body  given  pathogenic 
properties. 

The  possibility  of  making  any  thoroughly  satisfac- 
tory classification  is  rendered  still  more  difficult  by  the 
fact  that  many  necessarily  imperfect  attempts  have 
already  been  made,  so  that  there  is  a  great  deal  of  con- 
fusion, which  is  steadily  increased  as  new  varieties  are 
found  or  old  ones  reinvestigated  and  classified  differ- 
ently in  the  different  systems. 

As  one  of  the  more  successful  attempts  to  classify 
bacteria,  the  system  devised  by  Migula  is  here  given, 
simply  as  an  example.  The  morphology  of  bacteria  is 
used  as  the  basis  of  the  divisions  : 

FAMILIES. 

I.  Cells  globose  in  a  free  state,  not  elongat- 
ing in  any  direction  before  division 
into  1,  2,  or  3  planes  .  .  .  1.  Coccacese. 
II.  Cells  cylindrical,  longer  or  shorter, 
and  only  dividing  in  one  plane,  and 
elongating  to  twice  the  normal  length 
before  the  division. 

(1)  Cells  straight,  rod-shaped,  without 
sheath,   non-motile,    or    motile    by 

means  of  flagella    .         .         .         .2.  Bacteriaceae. 

( 2 )  Cells  crooked,  without  sheath         .  3.  Spirillacese. 

(3)  Cells  enclosed  in  a  sheath       .         .   4.  Chlamydobacteriace.'e. 

(4)  Cells  destitute  of  a  sheath,  united 
into  threads,  motile  by  means  of  an 

undulating  membrane    .         .         .5.  Beggiatoaceae. 


THE  CLASSIFICATION  OF  BACTERIA.        259 

GENERA. 

1.   Coccacece. 
Cells  without  organs  of  motion. 

a.  Division  in  one  plane       .         .         .  1.  Streptococcus. 

b.  Division  in  two  planes      .         .         .2.  Micrococcus. 

c.  Division  in  three  planes  .         .         .3.  Sarcina. 
Cells  with  organs  of  motion. 

a.  Division  in  two  planes     .         .         .4.  Planococcus. 
6.  Division  in  three  planes  .         .         .  5.  Planosarcina. 

2.  Hacleriacece. 

Cells  without  organs  of  motion         .         .  1.  Bacterium. 
Cells  with  organs  of  motion  (flagella). 

a.  Flagella  distributed  over  the  whole 

body 2.  Bacillus. 

6.  Flagella  polar .         .         .         .         .3.  Pseudomonas. 

3.  Spirillacece. 
Cells  rigid,  not  snake-like  or  flexuous. 

a.  Cells  without  organs  of  motion         .   1.  Spirosoma. 
6.   Cells  with  organs  of  motion  (flagella). 

1.  Cells  with  1,  very  rarely  2  to  3 

polar  flagella  .         .         .         .2.  Microspira. 

2.  Cells  with  polar  flagella-tufts     .  3.  Spirillum. 
Cells  flexuous 4.   Spirochseta. 

4.   Chlamydobacteriacece. 
Cell  contents  without  granules  of  sulphur. 

a.  Cell  threads  unbranched. 

I.  Cell  division  always  only  in  one  plane  1.  Streptothrix. 
II.  Cell  division  in  three  planes  previous 
to  the  formation  of  conidia. 

1.  Cells  surrounded  by  a  very  delicate, 

scarcely  visible  sheath  (marine)     .  2.   Phragmidiothrix. 

2.  Sheath  clearly  visible  (in  fresh  water)  3.  Crenothrix. 

6.   Cell  threads  branched        .         .  4.  Cladothrix. 
Cell  contents  containing  sulphur  granules  5.  Thiothrix. 

5.  Seggiatoacea. 

Only   one  species  known    (Beggiatoa,    Trev.),    which   is  scarcely 
separable  from  Oscillana. 


260  BACTERIOLOGY. 

A  study  of  the  above  table  will  show  that  it  makes 
changes  in  the  genus  of  some  of  the  most  common 
bacteria,  as  in  the  restoration  of  the  old  genus  bacte- 
rium and  the  assigning  to  it  of  all  non-motile,  rod- 
shaped  organisms,  thus  altering  the  genus  of  some  of 
the  most  common  pathogenic  bacteria  from  bacillus  to 
bacterium.  Other  changes  are  seen  in  the  spirilla. 
Any  such  scheme  is  at  times  very  arbitrary  in  placing 
some  varieties  under  one  generic  division  and  others 
closely  allied  in  another.  It  has  also  the  objection, 
already  noted,  that  it  is  only  one  of  several  classifi- 
cations already  in  use,  and  until  some  authoritative 
body  agrees  on  some  one  it  is  almost  useless  in  such 
a  volume  as  this  to  change  the  usually  employed  names 
for  others  which  are,  perhaps,  intrinsically  somewhat 
better.  Another  important  reason  for  waiting  is  that 
with  the  increase  of  our  knowledge  we  are  constantly 
changing  the  position  of  different  bacteria.  Thus  such  a 
well-known  germ  as  the  tubercle  bacillus  is  now  found 
to  produce,  under  certain  conditions,  long  thread-like 
branching  forms,  so  that  it  ceases  to  be  under  the  clas- 
sification of  Migula  a  bacterium.  We  will,  therefore, 
simply  use  the  usual  nomenclature,  and  consider  to- 
gether, in  so  far  as  is  practicable,  certain  groups  of 
bacteria  whose  members  are  closely  allied  to  each  other 
in  some  one  or  more  important  directions. 

The  Permanence  of  Bacterial  Species.  When  we  come 
to  study  special  varieties  or  groups  of  bacteria,  such  as 
the  bacilli  which  produce  typhoid  fever,  diphtheria,  and 
tuberculosis,  it  is  of  great  importance  for  us  to  deter- 
mine, if  possible,  to  what  extent  the  peculiar  character- 
istics which  each  of  these  groups  of  bacteria  possess  are 
permanent  in  the  generations  which  develop  from  them. 


THE  CLASSIFICATION  OF  BACTERIA.        261 

We  can  hardly  imagine  that  the  multitude  of  bacte- 
rial varieties  which  now  exist  have  always  existed. 
The  probability  is  very  strong,  that  with  succeeding 
generations  and  changing  conditions  new  bacterial 
varieties  have  developed  with  new  characteristics. 

From  time  to  time  the  changing  conditions  under 
which  life  progressed  probably  exposed  certain  animals 
to  the  invasion  of  varieties  which  never  before  had 
gained  access  to  them.  If  the  bacteria  found  the  soil 
suitable,  and  also  some  means  of  transmission  to  other 
animals  equally  susceptible,  a  pathogenic  species  became 
established  which  at  first,  perhaps,  found  conditions 
only  occasionally  favorable  to  it,  but  later  became  more 
parasitic  in  its  characteristics.  Thus  in  some  such  way 
a  multitude  of  bacterial  groups  arose,  some  of  which 
accustomed  themselves  to  the  conditions  present  in  the 
soil,  others  to  those  in  fishes,  others  to  those  in  birds, 
and  others  still  to  those  in  man. 

These  are,  however,  theories — what  has  been  actually 
observed  in  the  few  years  during  which  bacteria  have 
been  studied  ?  In  this  short  time  the  pathogenic  spe- 
cies as  observed  in  disease  have  kept  practically  unal- 
tered. The  diphtheria  bacilli  are  the  same  to-day  as 
when  Loffler  discovered  them  in  1884,  and  the  dis- 
ease itself  is  evidently  the  same  as  history  shows  it  to 
have  been  before  the  time  of  Christ.  The  same  is  true 
for  tuberculosis,  smallpox,  hydrophobia,  leprosy,  etc. 
Under  practically  unchanged  conditions,  therefore,  as 
exist  in  the  bodies  of  men,  bacteria  which  have  once 
become  established  as  parasites  continue  so  long  as  they 
remain  to  retain  their  peculiar  (specific)  characteristics. 
Whether  new  disease  varieties,  such  as  the  influenza 
bacillus,  are  coming  into  existence  from  time  to  time,  is, 


262  BACTERIOLOGY. 

of  course,  a  possibility,  but  not  a  certainty.  The  one 
thing  we  can  probably  safely  assert  is  that  there  is  no 
probability  that  any  saprophytic  variety  now  existing 
can,  under  any  possibility,  develop  into  the  now  recog- 
nized varieties  of  pathogenic  bacteria.  It  is  almost 
impossible  to  conceive  that  any  such  variety  should 
start  with  the  same  characteristics  and  then  develop 
parasitic  tendencies  under  exactly  the  same  circum- 
stances as  those  varieties  which  now  produce  disease. 

Attenuation.  It  is  now  a  well-established  fact  that 
the  great  majority  of  parasitic  bacteria  can  be  so  altered 
by  change  of  conditions,  and  especially  by  being  sub- 
jected to  unfavorable  conditions,  that  they,  while  mor- 
phologically the  same,  lose  their  power  of  developing 
in  the  body  and  of  producing  specific  poisons.  When 
either  or  both  these  properties  are  partially  destroyed 
they  can  usually  be  redeveloped;  but  when  power  to 
produce  specific  toxins  is  absolutely  lost,  it  is,  so  far 
as  we  now  know,  lost  forever. 

The  recovery  of  toxin  production  is  brought  about 
by  developing  the  micro-organism  for  a  considerable 
length  of  time  under  the  conditions  best  suited  for  it. 
The  recovery  of  the  ability  to  grow  in  the  body  of  any 
animal  species  is  brought  about  by  causing  the  germ 
to  develop  in  a  series  of  such  animals  whose  resistance 
has  been  overcome  by  reducing  their  vitality  through 
poisons,  heat,  cold,  etc.  Another  method  is  to  ac- 
custom the  micro-organism  to  the  animal's  body  by 
letting  it  remain  surrounded  by  the  animal  fluids  as  it 
rests  in  a  pervious  capsule  in  the  peritoneal  cavity. 


CHAPTER   XVIII. 

BACILLUS  OF  TUBERCULOSIS  (KOCH'S  TUBERCLE 
BACILLUS). 

IT  was  a  common  belief  many  years  ago  in  some 
countries  (kingdom  of  Naples,  1782)  that  tuberculosis 
was  an  infectious  disease;  but  it  is  only  within  com- 
paratively recent  times  that  the  infectiousness  of  tuber- 
culosis has  become  an  established  fact  in  scientific 
medicine.  Villemin  (1868)  was  the  first  to  show  ex- 
perimentally that  tuberculosis  might  be  induced  in 
healthy  animals  and  man  by  inoculations  of  tubercu- 
lous material.  Others  attempted  to  microscopically 
demonstrate  the  origin  of  the  disease  (Ziirn,  Buhl, 
Klebs,  Toussaint,  etc.);  but  these  investigations, 
though  paving  the  way  to  the  discovery,  which  it 
remained  for  Robert  Koch  to  make,  proved  to  be  un- 
satisfactory and  incomplete.  The  announcement  of 
the  discovery  of  the  tubercle  bacillus  was  made  by 
Koch,  in  March,  1882,  at  a  meeting  of  the  Physiolog- 
ical Society  of  Berlin.  At  the  same  time  satisfactory 
experimental  evidence  was  presented  as  to  its  etiolog- 
ical  relation  to  tuberculosis  in  man  and  in  susceptible 
animals,  and  its  principal  biological  characters  were 
given.  An  innumerable  number  of  investigators  now 
followed  Koch  into  this  field,  but  their  observations 
served  only  to  confirm  his  original  discovery. 


264  BACTERIOLOGY. 

The  bacilli  are  found  in  the  sputum  of  persons  suf- 
fering from  pulmonary  or  laryngeal  tuberculosis,  either 
free  or  in  the  interior  of  pus-cells;  in  miliary  tubercles 
and  fresh  caseous  masses  in  the  lungs  and  elsewhere; 
in  recent  tuberculous  cavities  in  the  lungs;  in  tuber- 
culous glands,  joints,  bones,  mucous  membranes,  and 
skin  affections;  in  the  lungs  of  cattle  suffering  from 
pulmonary  tuberculosis,  and  in  tubercular  nodules, 
generally  in  animals  which  are  infected  naturally  or 
by  experimental  inoculations. 

Morphological  Characters.  The  tubercle  bacilli  are 
slender,  non-motile  rods  of  about  0.2/2  in  diameter  by 
1.5  to  4/*  in  length.  (Plate  L,  Figs.  1,  2,  and  3.) 
Commonly  they  occur  singly  or  in  pairs,  and  are  then 
usually  slightly  curved;  frequently  they  are  observed 
in  smaller  or  larger  bunches.  Under  exceptional  con- 
ditions branching  forms  are  observed.  In  stained 
preparations  there  are  often  seen  unstained  portions, 
which  have  been  improperly  thought  to  be  spores. 
From  two  to  six  of  these  unstained  spaces  may  some- 
times be  noticed  in  a  single  rod,  and  under  moderate 
magnification  may  give  to  the  bacilli  the  appearance 
of  short  chains  of  streptococci.  In  old  cultures  irregu- 
lar forms  may  be  obtained,  the  rods  being  occasionally 
swollen  at  one  end  or  presenting  lateral  projections. 

The  staining  peculiarities  of  this  bacillus  are  very 
important,  for  by  them  its  differentiation  and  recogni- 
tion in  microscopical  preparations  of  sputum,  etc.,  are 
rendered  possible.  It  does  not  readily  take  up  the 
ordinary  aniline  colors,  but  when  once  stained  it  is 
very  difficult  to  decolorize,  even  by  the  use  of  strong 
acids.  Koch  first  recognized  it  in  a  staining  prepara- 
tion to  which  an  alkali  had  been  added — a  solution  of 


PLATE  I. 


FIG.  i. 


FIG.  2. 


Tubercle  bacilli,  in  red. 
Strepto-bacilli,  in  blue. 

X  i  ioo  diameters. 


Tubercle  bacilli,  in  red. 
Tissue,  in  blue. 

X  i  ioo  diameters. 


FIG.  3. 


FIG.  4. 


Very  large  tubercle  bacilli. 

Cells  in  specimen  are  in  blue, 

while  bacilli  are  red. 

X  i  ioo  diameters. 


Short  smegma  bacilli. 
Bacilli  in  specimen  are  red, 
rest  of  material  in  blue. 

X  i  ioo  diameters. 


BACILLUS  OF  TUBERCULOSIS.  265 

methylene-blue  with  caustic  potash.  More  recently 
Ehrlich  devised  a  method  of  staining  which  proved 
to  be  better,  viz.,  the  use  of  a  solution  of  an  aniline 
color — fuchsin  or  methyl-violet — in  a  saturated  aqueous 
solution  of  aniline  oil  and  decolorization  of  other  bac- 
teria with  a  solution  of  a  mineral  acid,  to  be  followed 
by  a  contrast  stain,  such  as  methylene-blue.  (Plate 
I.,  Figs.  1  and  2.)  Various  modifications  of  Ehrlich's 
method  are  now  commonly  used.  The  carbol-fuchsin 
solution  of  Ziehl  is  largely  employed ;  it  has  the  advan- 
tage of  acting  quickly  and  keeping  well.  The  tubercle 
bacilli  can  be  demonstrated  also  by  Gram's  method  of 
staining,  but  this  is  not  recommended  for  general  use. 

Biological  Characters.  The  bacillus  tuberculosis  is  a 
parasitic,  aerobic,  non-motile  bacillus,  and  .grows  only 
at  a  temperature  of  about  37°  C.  It  has  been  assumed 
that  this  bacillus  is  capable  of  forming  spores.  The 
refractile  spaces,  however,  are  not  found  to  possess  the 
regular  shape  and  brilliancy  of  ordinary  spores,  nor  have 
they  any  greater  resisting  power  to  heat,  desiccation, 
etc.,  than  the  homogeneous  bacilli.  Exposure  to  60°  C. 
in  water  destroys  them  in  fifteen  minutes.  The  bacilli 
have,  however,  a  somewhat  greater  resisting  power  than 
most  other  pathogenic  bacteria,  since  frequently  the 
bacilli  resist  desiccation  at  the  ordinary  temperatures 
for  months;  many  bacilli  die,  however,  soon  after  dry- 
ing. Portions  of  the  lung  from  a  tuberculous  cow,  dried 
and  pulverized,  produced  tuberculosis  in  guinea-pigs  at 
the  end  of  102  days  (Cad&ic  and  Malet).  They  retain 
their  vitality  for  a  considerable  time  in  putrefying  ma- 
terial. Cold  has  no  effect  upon  them.  When  dry  the 
more  resistant  organisms  stand  dry  heat  at  100°  C.  for 
hours;  but  when  moist,  as  in  milk,  they  are  more  quickly 


266  BACTERIOLOGY. 

killed— viz.,  at  55°  C.  in  one  hour,  at  60°  C.  in  fifteen 
minutes,  at  65°  C.  in  fifteen  minutes,  at  70°  C.  in  ten 
minutes,  at  80°  C.  in  five  minutes,  and  at  95°  C.  in  one 
minute.  One  reason  why  they  appear  to  withstand  in 
milk  high  temperatures  for  a  longer  time  than  given  in 
the  above  figures  is,  as  pointed  out  by  Theobald  Smith, 
that  when  heated  in  a  test-tube  the  cream  which  rises 
on  heating  is  exposed  on  its  surface  to  a  lower  tem- 
perature than  the  rest  of  the  milk,  and  as  this  contains 
many  bacteria  some  of  them  are  exposed  to  less  heat 
than  those  in  the  rest  of  the  fluid  receive. 

The  resisting  power  of  this  bacillus  against  chemi- 
cal disinfectants  is  considerable,  but  not  as  great  as  it 
is  apt  to  appear,  for,  as  in  sputum,  the  bacillus  is 
usually  protected  by  mucus  or  cell  protoplasm  from 
penetration  by  the  germicidal  agent.  It  is  not  always 
destroyed  by  the  gastric  juice  in  the  stomach,  as  is 
shown  by  successful  infection  experiments  in  susceptible 
animals  by  feeding  them  with  tubercle  bacilli  (Baumgar- 
ten  and  others).  They  are  destroyed  in  sputum  in  six 
hours  or  less  by  the  addition  of  an  equal  quantity  of  a 
3  per  cent,  solution  of  carbolic  acid,  and  in  about  one 
hour  by  an  equal  amount  of  a  5  per  cent,  solution. 
Bichloride  of  mercury  is  unsuitable  for  the  disinfection 
of  sputum  unless  used  in  very  strong  solutions  (1  :  500). 
From  recent  experiments  by  Yersin  upon  pure  cultures 
of  the  bacillus  it  appears  that  tubercle  bacilli  were  killed 
by  a  5  per  cent,  solution  of  carbolic  acid  in  thirty  sec- 
onds; by  1  per  cent,  in  one  minute;  absolute  alcohol, 
five  minutes;  iodoform-ether,  1  per  cent.,  five  minutes; 
mercuric  chloride,  1  :  1000  solution,  ten  minutes.  Salt- 
ing and  smoking  are  said  not  to  destroy  the  virulence 
of  tuberculous  meat  (Forster). 


BACILLUS  OF  TUBERCULOSIS.  267 

The  tubercle  bacillus  when  exposed  to  direct  sunlight 
is  killed  in  from  a  few  minutes  to  several  hours,  accord- 
ing to  the  thickness  of  the  layer  and  the  season  of  the 
year;  it  is  also  usually  destroyed  by  diffuse  daylight  in 
from  five  to  seven  days  when  placed  near  a  window. 
This  fact  is  worthy  of  note,  as  it  has  an  important 
hygienic  bearing.  Thus,  tuberculous  sputum  expector- 
ated upon  sidewalks,  etc.,  being  exposed  to  the  action 
of  direct  sunlight,  will  in  many  cases,  especially  in 
summer,  be  disinfected  by  the  time  it  is  in  a  condi- 
tion to  be  carried  into  the  air  as  dust.  For  the  same 
reason,  consumptive  patients  should  occupy  light,  sunny 
rooms  and  live  as  much  as  possible  in  the  open  air  and 
exposed  to  the  action  of  direct  sunlight. 

The  tubercle  bacillus  is  a  strict  parasite — that  is  to 
say,  its  biological  characters  are  such  that  it  could 
scarcely  find  natural  conditions  outside  of  the  bodies 
of  living  animals  favorable  for  its  multiplication.  But 
it  has  been  noted  that  when  it  is  cultivated  for  a  time 
in  artificial  media  containing  glycerin  it  may  grow  on 
the  surface  of  plain  veal  or  chicken  bouillon,  in  which 
media  it  fails  to  develop  when  introduced  directly  from 
a  culture  originating  from  the  body  of  an  infected  ani- 
mal. This  would  indicate  the  possibility  of  its  acquir- 
ing the  ability  to  grow  as  a  saprophyte.  The  experi- 
ments of  Nutall  also  show  that  the  bacillus  may  multi- 
ply, under  favorable  conditions,  in  tuberculous  sputum 
outside  of  the  body.  Notwithstanding  these  facts, 
it  is  probable  that  the  growth  of  tubercle  bacillus  out- 
side of  the  living  bodies  of  man  and  animals  is  so 
slight  as  to  have  no  practical  importance  in  causing 
infection. 

On  account  of  their  slow  growth  and  the  special  con- 


268  BACTERIOLOGY. 

ditions  which  they  require,  tubercle  bacilli  cannot  be 
grown  in  pure  culture  by  the  plate  method  on  the 
ordinary  culture  media.  Koch  first  succeeded  in  culti- 
vating and  isolating  this  bacillus  on  coagulated  blood- 
serum,  which  he  inoculated  by  carefully  rubbing  the 
surface  with  sections  of  tuberculous  tissue  and  then 
leaving  the  culture,  protected  from  evaporation,  for 
several  weeks  in  the  incubator.  Roux  and  Nocard 
afterward  showed  that  the  bacilli  from  man  and  ani- 
mals occasionally  grow  on  nutrient  agar  to  which 
glycerin  has  been  added  in  the  proportion  of  5  per 
cent. 

Growth  on  Coagulated  Blood-serum.  On  this  medium, 
which  is  regularly  used  to  obtain  the  first  culture,  the 
growth  first  becomes  visible  at  the  end  of  ten  to  four- 
teen days  at  37°  C.,  and  at  the  end  of  three  to  four 
weeks  a  distinct  and  characteristic  development  has 
occurred.  Small,  grayish-white  points  and  scales  first 
appear  on  the  surface  of  the  medium.  As  development 
progresses  there  is  formed  an  irregular,  membranous- 
looking  layer.  When  a  tiny  piece  of  this  is  removed, 
placed  on  a  cover-glass  without  rubbing,  stained,  and 
then  observed  under  the  microscope  the  surface  growth 
presents  a  characteristic  appearance,  the  bacilli  being 
arranged  in  parallel  rows  of  variously  curved  figures. 

Owing  to  the  greater  facility  of  preparing  and  steril- 
izing glycerin-cigar,  and  the  more  rapid  and  abundant 
growth  of  the  bacilli,  which  have  become  accustomed 
to  growth  outside  the  body  on  this  medium,  it  is  now 
usually  employed  in  preference  to  blood-serum  for 
preserving  cultures.  The  development  at  the  end  of 
fourteen  to  twenty-one  days  is  more  abundant  than 
upon  blood-serum  after  several  weeks.  When  numer- 


BACILLUS  OF  TUBERCULOSIS.  269 

ous  bacilli  have  been  distributed  over  the  surface  of 
the  culture  medium,  a  rather  uniform,  thick,  white 
layer,  which  subsequently  requires  a  slightly  yellowish 
tint,  is  developed;  when  the  bacilli  sown  are  few  in 
number,  or  are  associated  in  scattered  groups,  separate 
colonies  are  developed,  which  acquire  considerable 
thickness  and  have  more  or  less  irregular  outlines. 

Growth  on  Peptonized  Veal  or  Beef  Broth  Containing 
5  per  cent,  of  Glycerin.  On  these  media  the  tubercle 
bacillus  also  grows  readily  if  a  very  fresh  thin  film  of 
growth  from  the  glycerin  agar  is  floated  on  the  surface. 
The  latter  of  these  media  is  used  for  the  development  of 
tuberculin.  The  small  piece  of  pellicle  removed  from 
the  previous  culture  continues  to  enlarge  while  it  floats 
on  the  surface  of  the  liquid,  and  in  the  course  of  three 
to  six  weeks  covers  it  wholly  as  a  single  film,  which  on 
agitation  is  easily  broken  up  and  then  settles  on  the 
bottom  of  the  flask,  where  it  ceases  to  develop  further. 
The  liquid  remains  clear,  containing  in  solution  the 
products  formed  by  the  growth  of  the  bacillus,  and  is 
really  a  dilute  crude  tuberculin.  A  practical  point  of 
importance,  if  a  quick  growth  is  desired,  is  to  remove 
for  the  new  cultures  a  portion  of  the  pellicle  of  a  grow- 
ing bouillon  culture,  which  is  very  thin  and  actively 
increasing. 

The  Obtaining  of  Cultures  of  the  Tubercle  Bacillus  from 
Sputa  and  Infected  Materials  for  Diagnostic  Purposes. 
As  this  is  a  matter  of  great  and  increasing  importance, 
we  will  consider  in  detail  the  methods  which  have 
been  successfully  employed.  Pure  cultures  can  be  ob- 
tained directly  from  tuberculous  material  ;  but  as 
it  is  so  difficult  to  get  rid  of  the  other  bacteria 
which  are  almost  always  present,  and  which  grow 


270  BACTERIOLOGY. 

much  more  rapidly  and  take  possession  of  the  medium 
before  the  tubercle  bacillus  has  had  time  to  form 
visible  colonies,  it  is  best,  unless  human  tissues  can 
be  obtained  free  from  other  infection,  first  to  inocu- 
late a  guinea-pig,  both  subcutaneously  and  intraperito- 
ueally,  with  the  *  sputum,  and  then  obtain  cultures 
from  the  animal  as  soon  as  the  tubercle  infection  has 
fully  developed.  From  acute  tuberculosis  in  man  in 
other  regions  than  the  lungs,  where  mixed  infection 
usually  exists,  direct  cultures  on  blood-serum  may  be 
made. 

The  animals  thus  inoculated  usually  die  at  the  end 
of  three  weeks  to  four  months.  It  is  better,  however, 
to  kill  a  guinea-pig  which  by  its  enlarged  glands  shows 
evidence  of  tuberculosis,  and  to  remove,  with  the  greatest 
care  as  to  cleanliness,  one  or  more  nodules  from  the 
lungs,  spleen,  or  lymphatic  glands.  Animals  which 
develop  tuberculosis  acutely  are  apt  to  have  abundant 
tubercle  bacilli  and  give  successful  cultures,  while  the 
chronic  cases  usually  have  few  bacilli  and  give  unsuc- 
cessful cultures.  The  animals  after  being  killed  are 
placed  in  trays,  and  after  washing  with  a  5  per  cent, 
solution  of  carbolic  acid,  immediately  autopsied.  The 
skin  over  the  anterior  portion  of  the  body  having  been 
carefully  turned  back,  an  opening  is  cut  with  a  fresh 
set  of  sterile  instruments  into  the  thoracic  or  abdominal 
cavity;  then  with  a  sterile  forceps  the  lymph-gland 
portion  of  spleen  or  other  part  which  it  is  desired  to 
examine  is  removed  to  a  sterile  covered  beaker.  This 
tissue  if  suitable  may  be  sliced  in  thin  sections  and  con- 
veyed directly  to  the  surface  of  the  solid  culture  medium 
and  gently  rubbed  over  the  surface,  and  then  left  on  it, 
or  a  part  of  it  may  first  be  crushed  between  two  sterilized 


BACILLUS  OF  TUBERCULOSIS.  271 

glass  slides  and  then  transferred  to  the  serum  and 
rubbed  gently  over  its  surface.  Owing  to  the  liability 
of  the  blood -serum  to  become  too  dry  for  the  develop- 
ment of  the  bacillus,  it  is  necessary  to  keep  the  cul- 
ture moist  by  sealing  the  end  in  some  way,  as  by 
applying  a  rubber  cap  over  the  open  end  of  the  test- 
tube,  which  prevents  evaporation.  This  cap  should  be 
sterilized  in  a  solution  of  mercuric  bichloride  (1  : 1000) 
and  the  end  of  the  cotton  plug  burned  off  just  before 
applying  it,  to  destroy  any  spores  of  mould  fungi 
present.  Theobald  Smith,  who  has  had  a  very  large 
experience  in  growing  the  tubercle  bacillus,  gives  the 
following  details  as  to  his  method  : 

"Throughout  the  work  solidified  dog's  serum  was 
used.  The  dog  was  bJed  under  chloroform  and  the 
blood  drawn  from  a  femoral  artery,  under  aseptic 
conditions,  through  sterile  tubes  directly  into  sterile 
flasks.  The  serum  was  drawn  from  the  clot  with 
sterile  pipettes,  and  either  distributed  at  once  into 
tubes  or  else  stored  with  0.25  to  0.3  per  cent,  chloro- 
form added.  The  temperature  required  to  produce  a 
sufficiently  firm  and  yet  not  too  hard  and  dry  serum  is, 
for  the  dog,  75°  to  76°  C.;  for  horse  and  beef  serum 
it  is  from  4°  to  5°  lower.  The  tubes  containing  the 
serum  were  set  in  a  thermostat,  into  which  a  dish  of 
water  was  placed,  to  forestall  any  abstraction  of  moist- 
ure from  the  serum.  About  three  hours  suffice  for  the 
coagulation.  This  procedure  dispenses  with  all  sterili- 
zation excepting  that  going  on  during  the  coagulation 
of  the  serum.  It  prevents  the  gradual  formation  of 
membranes  of  salts,  which,  remaining  on  the  surface 
during  coagulation,  form  a  film  unsuited  for  bacteria. 
Tubes  of  coagulated  serum  should  be  kept  in  a  cold, 


272  BACTERIOLOGY. 

closed  space,  where  the  opportunities  for  evaporation 
are  slight.     They  should  always  be  kept  inclined. 

<(  The  ordinary  cotton-plugged  test-tubes  I  do  not 
use,  because  of  the  rapid  drying  out  permitted  by 
them  as  well  as  the  opportunities  for  infection  with 
fungi.  Instead,  a  tube  is  used  which  has  a  ground- 
glass  cap  fitted  over  it.  This  cap  contracts  into  a 
narrow  tube  plugged  with  glass-wool;  this  plug  is  not 
disturbed.  The  tube  is  cleaned,  filled,  and  inoculated 
by  removing  the  cap.  With  sufficient  opportunity  for 
the  interchange  of  air  very  little  evaporation  takes  place, 
and  contamination  of  the  culture  is  a  very  rare  occur- 
rence. In  inoculating  these  tubes  bits  of  tissue  which 
include  tuberculous  foci,  especially  the  most  recent,  are 
torn  from  the  organs  and  transferred  to  the  serum. 
Very  little  crushing,  if  any,  is  desirable  or  necessary. 
I  think  many  failures  are  due  to  the  often  futile 
attempts  to  break  up  firm  tubercles.  Nor  should  the 
bits  of  tissue  be  rubbed  into  the  surface,  as  is  some- 
times recommended.  After  a  stay  of  several  weeks  in 
the  thermostat  I  usually  remove  the  tubes  and  stir 
about  the  bits  of  tissue.  This  frequently  is  the  occa- 
sion for  a  prompt  appearance  of  growth  within  a  week, 
as  it  seems  to  put  certain  still  microscopical  colonies  in 
or  around  the  tissues  into  better  condition  for  further 
development.  The  thermostat  should  be  fairly  constant, 
as  urged  by  Koch  in  his  classic  monograph;  but  I  look 
upon  moisture  as  of  more  importance.  If  possible  a 
thermostat  should  be  used  which  is  opened  only  occa- 
sionally. Into  this  a  large  dish  of  water  is  placed, 
which  keeps  the  space  saturated.  Ventilation  should 
be  restricted  to  a  minimum.  As  a  consequence,  moulds 
grow  luxuriantly,  and  even  the  gummed  labels  must  be 


BACILLUS  OF  TUBERCULOSIS.  273 

replaced  by  pieces  of  stiff  manila  paper  fastened  to  the 
tube  with  a  rubber  band.  By  keeping  the  tubes  in- 
clined no  undue  amount  of  condensation  of  water  can 
collect  in  the  bottom,  and  the  upper  portion  of  the 
serum  remains  moist.  The  only  precaution  to  be 
applied  to  prevent  infection  with  moulds  is  to  thor- 
oughly flame  the  joint  between  the  tube  and  cap,  as 
well  as  the  plugged  end,  before  opening  the  tube." 

At  the  Saranac  Laboratory  beef  blood-serum  is  used 
in  ordinary  test-tubes,  which  are  sealed  by  rubber  caps. 
The  tubercles  are  crushed  between  sterile  glass  slides 
and  rubbed  gently  upon  the  serum  surface.  The  serum 
itself  must  not  be  too  firm.  It  should  just  be  solid 
enough  to  stand  upright.  The  results  thus  obtained 
by  Trudeau  and  Baldwin  have  been  as  good  as  those 
reported  by  Smith.  In  our  experience  all  methods  fre- 
quently fail  with  those  unfamiliar  with  them,  especially 
when,  as  shown  by  microscopical  examination,  the 
tubercular  tissue  used  contains  very  few  bacilli. 

Pathogenesis.  The  tubercle  bacillus  is  pathogenic  not 
only  to  man,  but  to  a  large  number  of  animals,  such  as 
the  monkey,  pig,  cow,  etc.  Guinea-pigs  are  extremely 
susceptible,  and  are  much  used  for  the  detection  of 
tubercle  bacilli  in  suspected  material.  When  inoculated 
with  the  minutest  doses  of  the  living  bacilli  they 
usually  succumb  to  the  disease.  Infection  is  most 
rapidly  produced  by  intraperitoneal  injection.  If  a 
large  dose  is  given  death  follows  in  from  ten  to 
twenty  days.  The  omentum  is  found  to  be  clumped 
together  in  sausage-like  masses  and  converted  into  hard 
knots,  which  contain  many  bacilli.  There  is  no  serous 
fluid  in  the  peritoneal  cavity,  but  generally  in  both 
pleural  sacs.  The  spleen  is  enlarged,  and  it,  as  well 

18 


274  BACTERIOLOGY. 

as  the  liver  and  peritoneum,  contains  large  numbers 
of  tubercle  bacilli.  If  smaller  doses  are  given  the 
disease  is  prolonged.  The  peritoneum  and  interior  or- 
gans— spleen,  liver,  etc. — are  then  filled  with  tubercles. 
On  subcutaneous  injection,  for  instance,  into  the  ab- 
dominal wall,  there  is  a  thickening  of  the  tissues  about 
the  point  of  inoculation,  which  breaks  down  in  about  a 
week  and  leaves  a  sluggish  ulcer  covered  with  cheesy 
material.  The  neighboring  lymph-glands  are  swollen, 
and  at  the  end  of  two  or  three  weeks  may  attain  the 
size  of  hazel-nuts.  Soon  an  irregular  fever  is  set  up, 
and  the  animal  becomes  emaciated,  usually  dying  within 
four  to  eight  weeks.  If  the  injected  material  contained 
only  a  small  number  of  bacilli  the  wound  at  the  point 
of  inoculation  may  heal  up  and  death  be  postponed  for 
a  long  time.  On  autopsy  the  lymphatic  glands  are 
found  to  have  undergone  cheesy  degeneration ;  the 
spleen  is  very  much  enlarged,  and  throughout  its  sub- 
stance, which  is  colored  dark  red,  are  distributed  masses 
of  nodules.  The  liver  is  also  enormously  increased  in 
size,  streaked  brown  and  yellow,  and  the  lungs  are  filled 
with  grayish-white  tubercles;  but,  as  a  rule,  the  kidneys 
contain  no  nodules.  Tubercle  bacilli  are  always  found 
in  the  affected  tissues,  but  the  more  chronic  the  process 
the  fewer  the  bacilli  that  are  apt  to  be  present. 

Rabbits  are  also  quite  susceptible  to  tuberculosis,  but 
considerably  less  so  than  guinea-pigs.  In  rabbits  death 
almost  invariably  follows  inoculations  of  tuberculous 
material  into  the  anterior  chamber  of  the  eye.  The 
local  effects  are  iris-tuberculosis  and  cheesy  degeneration 
of  the  pupil.  The  bacilli  then  penetrate  to  the  neigh- 
boring lymph-glands,  producing  softening  of  these,  then 
pulmonary  tuberculosis,  general  tuberculosis,  and  finally 


BACILLUS  OF  TUBERCULOSIS.  275 

death  at  the  end  of  several  weeks  or  months.  Subcuta- 
neous inoculations  are  less  effective,  and  in  small  doses 
do  not  always  kill.  Intravenous  and  intraperitoneal 
iujections  usually  produce  general  tuberculosis  and 
death  at  the  end  of  a  few  weeks.  The  tubercles  in 
rabbits  are  smaller,  as  a  rule,  and  the  spleen  and  liver 
not  so  much  enlarged  as  in  guinea-pigs,  but  the  kidneys 
not  infrequently  contain  nodules  of  the  size  of  a  pea. 

Of  other  susceptible  animals,  field-mice  and  cats 
are  readily  infected  by  artificial  inoculations  of  tuber- 
culous material  ;  rats,  white  mice,  and  dogs  only 
when  very  large  doses  are  given.  All  these  animals 
present  the  anatomical  lesions  of  miliary  tuberculosis. 
Bollinger  has  produced  intestinal  tuberculosis  in  calves 
by  inoculating  them  with  material  taken  from  a  tuber- 
culous man.  Canaries  are  also  susceptible  to  inocula- 
tions of  the  tubercle  bacillus;  but  not  sparrows.  Cold- 
blooded animals  of  various  kinds,  according  to  the 
experiments  of  Koch,  are  immune,  unless,  as  recently 
demonstrated,  the  bacilli  are  first  slowly  accustomed 
to  growth  at  low  temperatures.  Fowls  and  pigeons 
are  only  slightly  susceptible  to  the  bacillus  derived 
from  man.  Among  the  larger  birds,  parrots  alone 
would  seem  to  be  clearly  susceptible. 

Beside  the  artificial  modes  of  infection  already  re- 
ferred to,  tuberculosis  may  be  caused  in  animals  by 
feeding  them  with  tuberculous  material.  In  this  case 
evidence  of  infection  is  usually  shown  in  the  mesenteric 
glands  before  the  intestinal  walls  are  affected.  Zagari 
records  some  experiments  in  which  tubercle  bacilli  fed 
to  dogs  (one  of  the  less  susceptible  animals)  were  ab- 
sorbed by  the  mucous  membranes  of  the  intestines,  and 
thus  reached  the  internal  organs  without  producing  any 


276  BACTERIOLOGY. 

local  lesions  whatever.  It  would  seem  to  be  possible, 
therefore,  that  tubercular  infection  may  be  caused,  under 
certain  conditions,  by  absorption  through  serous  or 
mucous  membranes  without  the  evidence  of  any  local 
lesion. 

The  experimental  production  of  tuberculosis  by  in- 
halation of  bacilli  has  been  demonstrated  by  Koch  in 
guinea-pigs,  rabbits,  rats,  and  mice,  and  his  results  have 
since  been  confirmed  by  many  others;  but  in  these  ex- 
periments the  bacilli  were  usually  inhaled  in  the  form 
of  a  very  thin  spray  in  which  they  were  suspended. 
The  experimental  inhalation  of  dry  tubercular  dust  has 
seldom  proved  successful. 

Various  other  tubercular  affections  which  are  natural 
in  man  have  been  produced  experimentally  in  animals, 
as,  for  instance,  tuberculosis  of  the  joints  (Pawlowsky), 
tubercular  abscess  (Courmont),  etc. 

It  need  hardly  be  said  that  the  discovery  of  the 
tubercle  bacillus  has  elucidated  the  etiology  of  many 
diseases  the  origin  of  which  was  formerly  doubtful. 
Among  these  may  be  mentioned  the  various  forms 
of  tuberculosis  of  the  lungs  and  other  organs,  lupus, 
scrofula,  fungoid  inflammations  of  the  bones  and  joints, 
tuberculosis  in  cattle,  monkeys,  horses,  swine,  sheep, 
goats,  and  the  so-called  spontaneous  tuberculosis  in 
guinea-pigs  and  rabbits  in  cages  in  which  healthy  and 
artificially  infected  animals  have  been  kept  together. 

Of  domestic  animals  cattle  are  by  far  the  most  fre- 
quently attacked  by  this  disease.  It  is  also  not  uncom- 
mon in  young  swine.  Monkeys,  when  they  are  kept  in 
confinement,  die  almost  invariably  from  tuberculosis. 
Among  other  domestic  and  wild  animals  it  is  a  com- 
paratively rare  disease.  Birds,  with  the  exception  of 


BACILLUS  OF  TUBERCULOSIS.  277 

parrots,  are  not  subject  to  tuberculosis,  and  cold-blooded 
animals  are  altogether  immune. 

Beside  the  affections  already  referred  to  in  man  the 
following  diseases  have  been  traced  to  tubercular  origin: 
Among  skin  diseases,  so-called  inoculation-lupus,  tuber- 
culosis-verrucosa  cntis,  and  scrofuloderma;  choroidal 
tuberculosis,  idiopathic  serous  pleurisy  and  lymphatic 
enlargements  simulating  pseudoleuksemia. 

The  Action  upon  the  Tissues  of  the  Poisons  Produced 
by  the  Tubercle  Bacillus.  Soon  after  the  introduction 
into  the  tissues  of  tubercle  bacilli,  either  living  or  dead, 
the  cells  surrounding  them  begin  to  show  that  some 
irritant  is  acting  upon  them.  The  connective-tissue 
cells  become  swollen  and  undergo  mitotic  division,  the 
resultant  cells  being  distinguished  by  their  large  size 
and  pale  nuclei.  A  small  focus  of  proliferated  epithe- 
lioid  cells  is  thus  formed  about  the  bacilli,  and  accord- 
ing to  the  intensity  of  the  inflammation  these  cells  are 
surrounded  by  a  larger  or  smaller  number  of  the  lym- 
phoid  cells.  When  living  bacilli  are  present  and  multi- 
plying, the  lesions  progress,  the  central  cells  degenerate 
and  die,  and  a  cheesy  mass  results,  which  later  may  lead 
to  the  formation  of  cavities.  Dead  bacilli,  on  the  other 
hand,  give  off  sufficient  poison  to  cause  the  less  marked 
changes  only,  and  never  produce  cavities  (Prudden  and 
Hodenpyl).  Of  the  gross  pathological  lesions  produced 
in  man  by  the  tubercle  bacilli  the  most  characteristic 
are  small  nodules,  called  miliary  tubercles.  When 
young,  and  before  they  have  undergone  degeneration, 
these  tubercles  are  gray  and  translucent  in  color,  some- 
what smaller  than  a  millet-seed  in  size,  and  hard  in 
consistence. 

But  miliary  tubercles  are  not  the  sole  tuberculous 


278  BACTERIOLOGY. 

products.  The  tubercle  bacilli  may  cause  the  diffuse 
growth  of  a  tissue  identical  in  structure  with  that  of 
miliary  tubercles — that  is,  composed  of  a  basement 
substance  containing  epithelioid,  giant,  and  lymphoid 
cells.  This  diffuse  tubercle-tissue  also  tends  to  undergo 
cheesy  degeneration. 

Distribution  of  Tubercle  Bacilli  in  the  Tissues.  In 
acute  tuberculosis,  especially  when  caseation  is  rapidly 
spreading,  the  bacilli  are  usually  abundant.  They  are 
generally  scattered  irregularly  through  the  tissues  or 
in  small  groups.  They  are  occasionally  found  in  the 
leucocytes  and  in  the  giant  and  epithelioid  cells.  In 
subacute  and  chronic  lesions  they  are  usually  few  in 
number.  Sometimes  in  old  caseous  materials  numerous 
stained  granular  points  are  seen  ;  these  are  supposed 
by  some  to  be  a  resting  stage  similar  to  spores. 

Infection.  Infection  by  the  tubercle  bacillus  takes 
place  usually  through  the  respiratory  tract  or  the  diges- 
tive tract,  more  rarely  through  wounds  of  the  skin. 

In  the  majority  of  cases  the  mode  of  infection  is 
evident.  Pulmonary  tuberculosis  as  a  primary  dis- 
ease, and  not  occurring  in  young  children,  may  be  con- 
sidered to  be  caused  chiefly  by  the  direct  transmission 
of  tubercle  bacilli  through  kissing,  soiled  hands,  hand- 
kerchiefs, etc.,  or  by  the  inhalation  of  tuberculous  dust. 
Intestinal  and  mesenteric  tuberculosis,  which  is  rare 
among  adults  and  common  with  children,  is  probably 
due  not  only  to  swallowing  the  bacilli  received  in  the 
above  ways,  but  also  to  the  ingestion  of  tuberculous 
milk.  Lupus  is  probably  always  produced  by  the 
inoculation  of  tubercle  bacilli  on  the  skin  or  mucous 
membranes,  which  is  indicated  by  the  fact  that  the 
original  seat  of  the  disease  is  so  often  on  a  wounded 


BACILLUS  OF  TUBERCULOSIS.  279 

surface.     Localized  skin  tuberculosis  is  sometimes  pro- 
duced by  inoculation  at  autopsies. 

Infection  by  Inhalation  of  Tuberculous  Dust.  Cer- 
tainly one  of  the  common  modes  of  infection  is  by 
means  of  tuberculous  sputum,  which,  being  coughed 
up  by  consumptives  and  carelessly  expectorated,  dries 
and  distributes  numerous  virulent  bacilli  in  the  dust. 
As  long  as  the  sputum  remains  moist  there  is  no 
danger  of  dust  infection,  but  only  of  direct  contact; 
it  is  only  when  it  becomes  dry,  as  on  handkerchiefs, 
bedclothes,  and  the  floor,  etc.,  that  the  dust  is  a 
source  of  danger  for  infection.  A  great  number  of 
the  expectorated  and  dried  tubercle  bacilli  undoubtedly 
die,  especially  when  exposed  to  the  action  of  direct 
sunlight;  but  when  it  is  considered  that  from  one-half 
to  three  billion  virulent  tubercle  bacilli  (according  to 
the  experiments  of  Nutall)  may  be  expectorated  by  a 
single  tuberculous  individual  in  twenty-four  hours,  it 
is  evident  that  even  a  much  smaller  proportion  than  are 
known  to  stay  alive  will  suffice  in  the  immediate 
vicinity  of  consumptives  to  produce  infection  unless 
precautions  are  taken  to  prevent  it.  The  danger 
of  infection  is  greatest,  of  course,  in  the  close  neigh- 
borhood of  tuberculous  patients  who  expectorate  pro- 
fusely and  indiscriminately — that  is,  without  taking 
the  necessary  means  for  preventing  infection.  There 
is  comparatively  little  danger  of  infection  at  a  distance, 
as  in  the  streets,  for  instance,  where  the  tubercle  bacilli, 
even  if  present  in  the  dust,  have  become  so  diluted  that 
they  are  not  much  to  be  feared.  Exhaustive  experi- 
ments made  by  many  observers  have  shown  that  parti- 
cles of  dust  collected  from  the  immediate  neighborhood 
of  consumptives,  when  inoculated  into  guinea-pigs, 


280  BACTERIOLOGY. 

produced  tuberculosis  in  a  considerable  percentage  of 
them;  whereas  the  dust  from  rooms  inhabited  by  healthy 
persons  or  the  dust  of  the  streets  did  so  only  in  an  ex- 
tremely small  percentage.  Fliigge  is  probably  right 
in  thinking  that  the  dust  which  is  fine  enough  to 
remain  for  a  long  time  in  suspension  in  the  air  is  prac- 
tically free  from  living  pathogenic  bacteria.  It  is  the 
coarser  particles  in  which  the  bacilli  are  protected 
by  an  envelope  of  mucus  that  resist  drying  for  consider- 
able periods.  These  are  carried  only  short  distances 
by  air  currents.  Such  results  as  those  obtained  by 
Straus,  who,  examining  the  nasal  secretions  of  twenty- 
nine  healthy  persons  living  in  a  hospital  with  con- 
sumptive patients,  found  tubercle  bacilli  in  nine  of 
them,  must  be  accepted  with  some  reserve,  since  we 
know  that  in  the  air  there  are  bacilli  derived  from 
grasses  which  look  and  stain  like  tubercle  bacilli  and 
yet  are  totally  different.  It  has  been  argued  by  some, 
from  the  fact  that  about  one-seventh  of  all  men  die  from 
tuberculosis,  that  the  tubercle  bacilli  must  be  ubiquitous, 
and  that  precautions  are  useless;  but,  as  Cornet  has 
pointed  out,  this  does  not  mean  that  one-seventh  of  all 
men  living  are  tuberculous,  for  no  man  is  tuberculous 
during  the  entire  course  of  his  life,  but  only  for  a  lim- 
ited period  (variously  estimated  at  from  three  to  eight 
years).  It  may,  therefore,  be  said  that  the  danger  of 
infection  from  tuberculosis  in  general  is  not  so  great 
after  all,  but  that  on  this  account  it  is  all  the  more  to 
be  feared  and  guarded  against  in  the  immediate  neigh- 
borhood of  consumptives.  Those  who  are  most  liable 
to  infection  from  this  source  are  the  families,  the  nurses, 
the  fellow-workmen,  and  fellow-prisoners  of  persons 
suffering  from  the  disease.  In  this  connection,  also, 


BACILLUS  OF  TUBERCULOSIS.  281 

attention  may  be  drawn  to  the  fact  that  rooms  which 
have  been  recently  occupied  by  consumptives  are  not 
infrequently  the  means  of  producing  infection  (as  has 
been  clinically  and  experimentally  demonstrated)  from 
the  deposition  of  tuberculous  dust  on  furniture,  walls, 
floors,  etc.  Fliigge  has  recently  drawn  attention  to  the 
fact  that  in  coughing,  sneezing,  etc.,  very  fine  parti- 
cles of  throat  secretion  are  thrown  out  and  carried  by 
air  currents  many  feet  from  the  patient  and  remain 
suspended  in  the  air  for  a  considerable  time.  This 
is  another  means  of  infection,  but  probably  an  in- 
frequent one.  We  have  now  to  encourage  us  a  mass 
of  facts  which  go  to  show  that  when  the  sputa  is  care- 
fully looked  after  there  is  very  little  danger  of  the 
infection  of  others  except  by  close  personal  contact. 

Individual  Susceptibility.  It  is  believed  by  many 
that  in  demonstrating  the  possibility  of  infection  in 
pulmonary  tuberculosis  its  occurrence  is  sufficiently 
explained;  but  they  leave  out  another  and  most  impor- 
tant factor  in  the  production  of  an  infectious  disease 
— individual  susceptibility.  That  this  susceptibility, 
or  "  predisposition/ '  as  it  is  improperly  called,  may 
be  either  inherited  or  acquired  is  now  an  accepted 
fact  in  medicine.  It  is  even  thought  that  the  phys- 
ical signs  and  characters — the  phthisical  habit — which 
indicate  this  susceptibility  can  be  externally  recog- 
nized. Whatever  may  be  the  opinion  with  regard  to 
these  outward  signs,  there  is  no  doubt  that  personal 
susceptibility  is  of  the  greatest  importance  in  the  pro- 
duction of  this  disease.  Unquestionably,  vast  differ- 
ences exist  in  different  individuals  in  the  intensity 
of  the  tubercular  process  in  the  lung.  That  this  does 
riot  depend  chiefly  upon  a  difference  in  virulence  of 


282  BACTERIOLOGY. 

the  infection  is  evident,  from  the  fact  that  individuals 
contracting  tuberculosis  from  the  same  source  are  at- 
tacked with  different  severity,  and  that  there  is,  as  a 
rule,  no  great  difference  in  degrees  of  virulence  in  the 
tubercle  bacilli  obtained  from  different  sources.  As  is 
seen  from  the  results  of  post-mortem  examinations  in 
which  the  remains  of  old  tubercular  processes  have  been 
found  in  the  lungs  of  about  one-third  of  all  the  bodies 
examined,  many  cases  of  pulmonary  phthisis  must  occur 
without  showing  any  visible  evidences  of  disease,  and 
heal  of  their  own  accord.  The  possibility  of  favorably 
influencing  in  many  an  existing  tuberculosis  by  treat- 
ment also  proves  that,  under  natural  conditions,  there  is 
a  varying  susceptibility  to  the  disease.  Clinical  experi- 
ence teaches,  likewise,  that  poor  hygienic  conditions, 
depressing  surroundings  (as  in  asylums  and  prisons), 
obstinate  bronchial  affections,  diabetes,  and  other  ex- 
hausting diseases  increase  the  susceptibility  to  phthisis. 
Animal  experiments  have  shown  that  not  only  are  there 
differences  of  susceptibility  in  various  animal  species, 
but  also  an  individual  susceptibility  in  the  same  species. 
This  is  not  so  evident  among  guinea-pigs,  which  are 
so  susceptible  that  they  succumb  to  an  inoculation  of 
the  minutest  dose  of  virulent  bacilli;  but  rabbits  are 
not  always  killed  by  subcutaneous  inoculations,  though 
some  individuals  die  from  very  small  doses.  Dogs, 
rats,  and  other  more  resistant  animals  show  this  still 
more  plainly.  Man  cannot  be  placed  on  the  same  plane 
of  susceptibility  to  tuberculosis  with  guinea-pigs,  for 
with  him  the  disease  often  remains  local  or  is  entirely 
cured.  The  doctrine  of  individual  susceptibility,  there- 
fore, is  seen  to  be  founded  on  fact,  although  the  reasons 
for  it  are  only  partially  understood. 


BACILLUS  OF  TUBERCULOSIS.  283 

Infection  by  Ingestion  of  Milk  and  Meat.  Phthisi- 
cal sputum,  however,  is  not  held  responsible  for  the 
occurrence  of  all  human  tuberculosis.  Milk  also 
serves  as  a  conveyer  of  infection,  whether  it  be  the  milk 
of  nursing  mothers  suffering  from  consumption  or  the 
milk  of  tuberculous  cows.  The  transmission  of  tubercle 
bacilli  in  the  milk  of  tuberculous  individuals  has  only 
been  indirectly  established  in  human  beings,  but  in 
cow's  milk  it  has  been  abundantly  proved.  Formerly 
it  was  thought  that  in  order  to  produce  infection  by 
milk  there  must  be  local  tubercular  affection  of  the 
udder;  but  it  is  known  now  that  tubercle  bacilli  may 
be  found  in  the  milk  when  an  internal  organ  is  infected 
and  when  careful  search  fails  to  detect  any  udder  dis- 
ease. So  that  the  milk  of  every  cow  which  has  any 
internal  tubercular  infection  must  be  considered  as  pos- 
sibly containing  tubercle  bacilli.  Rabinowitsch  and 
Kempner  proved  beyond  all  question  that  not  only  the 
milk  of  tubercular  cattle  which  showed  no  appreciable 
udder  disease,  but  also  those  in  which  tuberculosis  was 
only  detected  through  tuberculin,  frequently  contained 
tubercle  bacilli.  Different  observers  have  found  tubercle 
bacilli  in  the  milk  of  from  20  to  60  per  cent,  of  tuber- 
culous cows.  When  we  consider  the  prevalence  of  tuber- 
culosis among  cattle  we  can  readily  realize,  if  the 
bovine  bacillus  readily  infects  human  beings,  the 
danger  to  which  children  are  exposed  from  this  source 
of  infection.  Thus,  taking  the  abattoir  statistics  of 
various  countries,  we  find  that  in  Prussia  8.3  per  cent, 
of  the  cattle  slaughtered  were  tuberculous;  in  Dresden, 
14.4  per  cent.;  in  London,  25  per  cent.;  in  Berlin,  12 
per  cent.;  in  New  York,  about  7  per  cent.  Another 
possible  source  of  infection  in  intestinal  tuberculosis  is 


284  BACTERIOLOGY. 

the  flesh  of  tuberculous  cattle.  Here  the  same  condi- 
tions hold  good  as  in  the  infection  by  milk,  only  the 
danger  is  considerably  less,  from  the  fact  that  meat  is 
usually  cooked,  and  also  because  the  muscular  tissues 
are  seldom  attacked.  In  view  of  the  great  mortality 
from  tubercular  diseases  among  mankind,  the  legisla- 
tive control  and  inspection  of  cattle  and  milk  would 
seem  to  be  an  absolute  necessity.  As  a  practical  and 
simple  method  of  preventing  infection,  especially 
among  children,  the  sterilization  (by  heat)  of  the  milk 
used  as  food  must  commend  itself  to  all.  It  is  only  right 
to  state,  however,  that  the  actual  proof  that  human  tuber- 
culosis has  come  from  milk  or  food  infected  with  bovine 
tuberculosis  is  very  small,  and  that  it  is  perfectly  pos- 
sible that  the  bovine  bacilli  may  not  be  as  virulent  for 
man  as  for  animals,  still  we  know  that  human  tubercu- 
losis produces  bovine  tuberculosis  in  young  and  suscep- 
tible animals,  and  the  reverse  is  in  all  probability  true. 
The  relation  of  bovine  to  human  tubercle  bacilli  will 
be  discussed  later  in  this  chapter. 

Auto-infection  by  Swallowing  Sputum.  The  secondary 
forms  of  tuberculosis  which  often  succeed  a  primary 
infection  of  the  lungs  may  be  explained  as  an  auto- 
infection  from  the  swallowing  of  sputum  containing 
bacilli,  these  passing  through  the  gastric  juice  unaf- 
fected. It  is  a  wonder,  indeed,  that  intestinal  tuber- 
culosis is  not  more  common  than  it  is  in  consumption; 
but  this  is  probably  due  to  the  fact  that  in  adults  the 
intestines  are  comparatively  insusceptible.  Tubercu- 
losis may  also  begin  as  a  local  infection  in  the  lungs  or 
intestines,  and  theiice  extend  to  other  parts  of  the  body, 
until,  passing  into  the  circulation,  a  general  miliary 
tuberculosis  results. 


BACILLUS  OF  TUBERCULOSIS.  285 

Hypothesis  of  Transmissibility  of  Tubercle   Bacilli  to 
the  Foetus.     Baumgarten  and  others  have  advanced  a 
hypothesis  to  account  for  certain  obscure  cases  of  tuber- 
culosis— namely,  that  of  the  transmissibility  of  tubercle 
bacilli  from  the  mother  to  the  unborn  babe.     There 
seems  to  be  some  evidence  of  the  possible  transmission 
of  tubercular  poison  from  the  mother  to  the  foetus  in 
animals.     The   first   authentic  case   recorded   is   that 
reported  by  Johne  of  an  eight-months-old  calf  foetus; 
other  cases  have  since  been  reported.     With  regard  to 
tuberculosis  in  the  human  foetus  the  evidence  is  not  so 
clear,  though  several  cases  have  been  reported  of  tuber- 
culosis in  very  young  babies  only  a  few  weeks  old,  and 
two  cases  are  recorded  of  placental  tuberculosis.     The 
fact  that  statistics  show  a  greater  frequency  of  tuber- 
cular diseases  in  children  during  the  first  than  in  the 
following  years  of  life  does  not  strengthen  the  hypo- 
thesis of  infection  in  utero ;  for  nursing  babies  would 
naturally  be  more  exposed  to  infection  through   the 
mother's  milk  and  through  personal  contact  than  others, 
and,  beside,  the  more  tender  the  life  of  the  infant  the  more 
susceptible  it  would  be  ordinarily  to  indirect  infection 
from  a  tuberculous  mother.    Experimental  proof,  how- 
ever, of  the  actual  transmission  of  tubercle  bacilli  from 
the  mother  to  the  foetus  in  animals  has  recently  been 
furnished.    De  Blenzi  found  that  in  five  out  of  eighteen 
cases  in  guinea-pigs  such  transmission  of   bacilli  did 
take  place,  and  Gartner  confirmed  the  same  in  numer- 
ous experiments  on  rabbits,  mice,  and  canaries.     The 
infection  resulted  not  only  from  animals  affected  with 
general  miliary  tuberculosis,  but  also  from  local  dis- 
ease of  the  lungs;  but  in  the  majority  of  cases  very 
few  bacilli  were   transmitted   to  the  foetus — so  few, 


286  BACTERIOLOGY. 

indeed,  that  it  required  the  inoculation  of  the  entire 
contents  of  the  body  to  cause  tuberculosis  in  guinea- 
pigs;  moreover,  only  one  or  two  of  a  litter  were  affected 
at  one  time.  According  to  these  experiments  one  would 
expect  to  find  in  man  foetal  or  placental  tubercular  in- 
fection more  common  than  it  is,  whereas  it  is  extremely 
rare,  even  if  the  few  cases  reported  be  accepted  as  proven. 
Possibly  the  few  bacilli  which  may  be  transmitted  to 
the  foetus  do  not  find  conditions  favorable  for  their 
development,  and,  being  so  few  in  number,  die;  or 
they  may  remain  latent,  as  has  been  suggested,  for 
certain  lengths  of  time  without  producing  visible  effects, 
and  only  show  symptoms  of  infection  later;  but  we  have 
no  experimental  confirmation  of  any  such  latency  ex- 
isting with  regard  to  the  tubercle  bacillus,  and  it  is  not 
to  be  assumed  that  it  does  exist.  As  to  the  infection  of 
the  foetus  from  the  paternal  side,  where  the  father  has 
tuberculosis  of  the  scrotum  or  seminal  vessels  (which 
have  been  found  to  be  tuberculous  in  exceptional  cases), 
we  have  no  reason  to  suppose  that  such  can  occur. 
There  are,  however,  some  grounds  for  belief  that  infec- 
tion in  this  way  may  take  place  from  husband  to  wife. 
Thus,  Gartner  found,  as  a  result  of  his  experiments  in 
animals,  that  a  large  majority  of  the  guinea-pigs  and 
rabbits  which  were  brought  together  with  males  whose 
semen  contained  tubercle  bacilli  died  of  primary  gen- 
ital tuberculosis;  but  from  the  rarity  of  this  affection 
in  women  and  cows  it  may  be  assumed  that  tubercle 
bacilli  occur  very  much  less  frequently  in  semen  of  men 
and  cattle  than  in  that  of  the  smaller  animals. 

Length  of  Time  Tubercle  Bacilli  Remain  Virulent  in 
Sputum.  Of  considerable  importance  in  studying  the 
subject  of  tubercular  infection  is  the  question  of  the 


BACILLUS  OF  TUBERCULOSIS.  287 

length  of  time  daring  which  the  tubercle  bacillus  re- 
tains its  virulence,  and  whether  there  are  any  naturally 
attenuated  varieties.  According  to  experimental  in- 
vestigations, the  virulence  of  dried  tubercular  sputum 
is  not  suddenly  but  gradually  lost,  a  certain  pro- 
portion of  it  retaining  its  specific  infective  power 
under  ordinary  conditions,  as  in  a  dwelling-room,  for 
at  least  two  or  three  months.  An  instance  is  reported 
by  Ducor  (Paris,  1890)  of  a  healthy  family  having 
been  infected  with  tuberculosis  from  living  in  a  room 
which  had  been  occupied  by  a  consumptive  two  years 
before,  and  on  examining  the  sputum-stained  wall-paper 
not  only  were  tubercle  bacilli  found  in  it,  but  upon  being 
inoculated  into  guinea-pigs  they  died  of  tuberculosis. 

Attenuation.  Metschnikoff  states  that  when  kept 
at  a  temperature  of  42°  C.  for  some  time  the  tubercle 
bacillus  undergoes  a  notable  diminution  in  its  patho- 
genic power,  and  that  when  kept  at  a  temperature  of 
43°  to  44°  C.  it  after  a  time  only  induces  a  local  abscess 
when  injected  subcutaneously  into  guinea-pigs.  The 
experiments  of  Lote  also  indicate  that  an  attenuation 
of  virulence  has  occurred  in  the  cultures  preserved  in 
Koch's  laboratory,  originating  in  1882  from  the  lungs 
of  a  tuberculous  ape.  A  culture  of  ours  which  we  ob- 
tained from  Trudeau,  and  which  has  grown  now  either 
at  Saranac  or  in  our  laboratory  for  six  years,  is  no 
longer  capable  of  causing  tuberculosis  in  guinea-pigs, 
although  originally  virulent. 

Mixed  Infection.  Some  time  ago  attention  was  drawn 
to  the  fact  that  tuberculosis,  whether  of  the  lungs, 
lymphatics,  or  cold  abscesses,  was  often  a  mixed  in- 
fection. The  other  micro-organisms  with  which  the 
tubercle  bacillus  is  most  commonly  associated  are  the 


288  BACTERIOLOGY. 

streptococcus,  pneumococcus,  and  influenza  bacillus. 
Besides  these  many  other  varieties  are  met  with  occa- 
sionally in  individual  cases.  What  the  influence  of 
this  secondary  or  mixed  infection  is,  under  all  circum- 
stances, is  not  exactly  known;  but  generally  the  effect 
is  an  unfavorable  one,  and  not  infrequently  on  their 
invasion  the  disease  takes  on  a  septicsemic  character. 
For  the  technique  employed  in  examining  sputa  for 
mixed  infection,  see  later  in  this  chapter. 

Immunization.  As  in  other  infectious  diseases,  many 
attempts  have  been  made  to  produce  an  artificial  immu- 
nity against  tuberculosis,  but  so  far  the  results  have 
been  unsatisfactory.  Among  the  numerous  medicinal 
agents  that  have  been  tried  to  protect  animals  against 
the  action  of  the  tubercle  bacillus  may  be  mentioned 
tannin,  menthol,  sulphuretted  hydrogen,  mercuric  chlo- 
ride, creosote,  creolin,  phenol,  arsenic,  eucalyptol,  etc. 
Various  inoculation  experiments  with  cultures  of  the 
tubercle  bacilli  and  their  products  have  been  made, 
and  though  the  results  reported  in  some  cases  have 
been  temporarily  favorable,  immunization  has  never 
been  satisfactorily  produced. 

Koch's  Tuberculin.  The  discovery  by  Koch  of  toxins 
in  cultures  of  the  tubercle  bacillus  which  possess  prop- 
erties which  explain  its  pathogenic  power  must  rank 
as  one  of  the  first  importance  in  scientific  medicine, 
on  account  of  what  it  has  led  up  to,  even  if — as  ap 
pears  probable — the  final  verdict  may  be  that  its  thera- 
peutic value  in  the  treatment  of  tubercular  diseases  in 
man  is  very  slight. 

Tuberculin  contains  all  the  products  of  the  growth 
of  the  tubercle  bacilli  in  the  nutrient  bouillon  as  well 
as  some  substances  extracted  from  the  bodies  of  the 


BACILLUS  OF  TUBERCULOSIS.  289 

bacilli  themselves.  It  also  contains  all  the  albuminoid 
and  other  materials  originally  contained  in  the  bouillon 
which  have  remained  unaffected  by  the  activities  of  the 
bacilli.  There  are  two  preparations  known  respec- 
tively as  the  old  and  the  new  tuberculin. 

Old  tuberculin  is  prepared  as  follows  :  The  tubercle 
bacillus  is  cultivated  in  an  infusion  of  calf  s  flesh,  or  of 
beef  flesh,  or  extract  to  which  1  per  cent,  of  peptone 
and  4  to  5  per  cent,  of  glycerin  have  been  added,  the 
culture  liquid  being  slightly  alkaline.  The  inoculation 
is  made  upon  the  surface  from  a  piece  of  very  thin 
pellicle  from  a  young  bouillon  culture,  or,  if  the  bou- 
illon culture  is  unobtainable,  with  small  masses  from  a 
culture  on  glycerin-agar.  These  masses,  floating  on 
the  surface,  give  rise  in  from  three  to  six  weeks,  accord- 
ing to  the  rapidity  with  which  the  culture  grows,  to  an 
abundant  development  and  to  the  formation  of  ,a  toler- 
ably thick  and  dry,  white  crumpled  layer,  which  finally 
covers  the  entire  surface.  At  the  end  of  four  to  eight 
weeks  development  ceases,  and  the  layer  after  a  time 
sinks  to  the  bottom.  Fully  developed  cultures,  after 
having  been  tested  for  purity  by  a  microscopical  exami- 
nation, are  passed  into  a  suitable  vessel  and  evaporated 
to  one-tenth  of  their  original  bulk  over  a  water-bath 
at  a  temperature  of  70°  to  80°  C.  The  liquid  is  then 
filtered  through  chemically  pure  sterilized  filter-paper. 
The  crude  tuberculin  thus  obtained  contains  40  to  50  per 
cent,  of  glycerin  and  keeps  well,  retaining  its  activity 
indefinitely. 

The  method  of  treatment  and  the  results  obtained 
from  the  old  tuberculin  have  been  described  recently 
by  Koch  briefly  as  follows  :  After  each  injection,  which 
should  be  large  enough  to  cause  a  slight  but  not  a  great 

19 


290  BACTERIOLOGY. 

rise  of  temperature,  a  noticeable  improvement  in  the 
tuberculous  process  results.  The  amount  of  tuberculin 
injected  is  continually  increased,  so  as  to  continue  the 
moderate  reactions.  After  several  months  all  reactions 
cease,  the  patients  having  become  temporarily  immune 
to  the  toxin,  but  not  to  the  growth  of  the  bacillus. 
Further  injections  are  now  useless  until  this  immunity 
has  passed.  During  the  treatment  the  bacilli  themselves 
have  not  been  directly  affected,  and  when  the  treatment 
is  interrupted  the  tuberculous  process  is  apt  to  progress. 
Many  cases,  however,  of  pure  tuberculosis  become  cured 
or  greatly  benefited  by  several  periods  of  treatment. 

The  substances  produced  in  the  body  by  the  old  tuber- 
culin neutralized  the  tubercular  toxins,  according  to 
Koch,  but  were  not  bactericidal.  After  a  series  of  ex- 
periments, he  considered  the  difficulty  to  be  due  to  the 
nature  of  the  envelope  of  the  tubercle  bacillus,  which 
made  it  difficult  to  obtain  the  substance  of  the  bacilli 
in  soluble  form  without  so  altering  it  by  heat  or  chem- 
icals that  it  was  useless  to  produce  immunizing  sub- 
stances. He  conceived  that  immunity  was  not  produced 
in  man  for  somewhat  similar  reasons — possibly,  the 
bacilli  never  giving  out  sufficient  toxin  to  cause  cura- 
tive substances  to  be  produced.  He  therefore  decided 
to  grind  up  the  dried  bacilli  and  soak  them  in  water, 
and  thus  obtain,  if  possible,  without  the  addition  of 
heat,  a  soluble  extract  of  the  body-substance  of  the 
bacilli,  which  he  hoped  would  be  immunizing.  He 
also  tried  to  eliminate  as  much  as  possible  of  the  toxic 
products  which  produce  fever.  Biichner  by  a  different 
method,  through  crushing  under  a  great  pressure 
tubercle  bacilli  mixed  with  sand,  and  thus  squeezing 
out  their  protoplasm,  obtained  a  very  similar  substance. 


BACILLUS  OF  TUBERCULOSIS.  291 

The  new  tuberculin  formed  by  either  of  these  methods 
is  a  watery  extract  of  the  soluble  portions  of  the  un- 
altered tubercle  bacilli.  As  can  be  readily  seen,  in  a 
preparation  thus  made  contamination  is  difficult  to 
avoid,  freedom  from  intact  bacilli  is  uncertain,  and  the 
strength  of  the  solution  prepared  at  different  times  is 
variable.  Twenty  per  cent,  of  glycerin  is  added  to 
preserve  the  tuberculin  from  contamination.  After 
three  years  of  trial  the  results  obtained  with  the  new 
tuberculin  preparations  cannot  be  considered  to  have 
exerted  either  very  different  or  very  superior  effects  to 
the  older  product. 

As  to  the  results  obtained  in  general  the  reports  are 
as  yet  conflicting.  Lupus  seems  to  be  decidedly  bene- 
fited for  a  time  both  by  the  old  and  the  new  tuberculin. 
Relapses  are,  however,  common.  On  advanced  phthisis, 
laryngeal  tuberculosis,  arid  other  tubercular  processes 
no  effects  have  been  noted,  and  nearly  every  one  dis- 
approves of  their  use  in  these  cases  as  well  as  in  those 
where  mixed  infection  is  suspected  ;  even  in  cases  of 
beginning  infection,  opinions,  as  a  whole,  are  not  very 
enthusiastic.  The  new  tuberculin  is,  except  when 
prepared  with  the  utmost  care, .a  dangerous  substance, 
for  Trudeau,  Baldwin  and  others  found  that  guinea-pigs 
injected  with  it  not  only  did  not  become  immunized, 
but  actually  became  infected  from  the  living  bacilli  in 
the  fluid. 

The  chief  use  to  which  tuberculin  has  been  put  is  as 
an  aid  to  the  diagnosis  of  tuberculosis  in  cattle  and 
human  beings,  and  for  this  purpose  it  has  proved  to  be 
of  inestimable  value.  Numerous  experiments  made  by 
veterinary  surgeons  show  that  the  injection  of  tuber- 
culin in  tuberculous  cows  in  doses  of  25  to  50  centi- 


292  BACTERIOLOGY. 

grammes  produces  in  at  least  95  per  cent,  a  rise  of  tem- 
perature of  from  1°  to  3°  C.  The  febrile  reaction  occurs 
in  from  twelve  to  fifteen  hours  after  the  injection.  Its 
intensity  and  duration  do  not  depend  upon  the  extent 
of  the  tuberculous  lesions,  but  is  even  more  marked 
when  these  are  slight  than  in  advanced  cases.  In  non- 
tuberculous  animals  no  reaction  occurs,  or  one  much 
less  than  in  tuberculous  animals,  and  the  results  ob- 
tained on  autopsy  justify  the  suspicion  that  tubercu- 
losis exists  if  an  elevation  of  temperature  of  a  degree 
or  more  occurs  from  the  subcutaneous  injection  of  the 
dose  mentioned.  For  these  injections  the  crude  old 
tuberculin  is  used,  which  for  the  convenience  of  admin- 
istration is  diluted  with  water.  The  following  are  the 
directions  for  inspecting  herds  for  tuberculosis  : 

(t  Inspections  should  be  carried  on  while  the  herd  is 
stabled.  If  it  is  necessary  to  stable  animals  under 
unusual  conditions  or  among  unusual  surroundings  that 
make  them  uneasy  and  excited  the  tuberculin  test  should 
be  postponed  until  the  cattle  have  become  accustomed 
to  the  conditions  they  are  subjected  to,  and  then  begin 
with  a  careful  physical  examination  of  each  animal. 
This  is  essential,  because  in  some  severe  cases  of  tuber- 
culosis, on  account  of  saturation  with  toxins,  no  reaction 
follows  the  injection  of  tuberculin,  but  experience  has 
shown  that  these  cases  can  be  discovered  by  physical 
examination.  This  should  include  a  careful  examina- 
tion of  the  udder  and  of  the  superficial  lymphatic  glands 
and  auscultation  of  the  lungs. 

"  Each  animal  should  be  numbered  or  described  in 
such  a  way  that  it  can  be  recognized  without  difficulty. 
It  is  well  to  number  the  stalls  with  chalk  and  transfer 
these  numbers  to  the  temperature-sheet,  so  that  the 


BACILLUS  OF  TUBERCULOSIS.  293 

temperature  of  each  animal  can  be  recorded  in  its 
appropriate  place  without  danger  of  confusion.  The 
following  procedure  has  been  used  extensively  and  has 
given  excellent  results  : 

"  (a)  Take  the  temperature  of  each  animal  to  be  tested 
at  least  twice,  at  intervals  of  three  hours,  before  tuber- 
culin is  injected. 

"  (b)  Inject  the  tuberculin  in  the  evening,  prefer- 
ably between  the  hours  of  six  and  nine.  The  injection 
should  be  made  with  a  carefully  sterilized  hypoder- 
matic syringe.  The  most  convenient  point  for  injec- 
tion is  back  of  the  left  scapula.  Prior  to  the  injection 
the  skin  should  be  washed  carefully  with  a  5  per  cent, 
solution  of  carbolic  acid  or  other  antiseptic. 

"  (c)  The  temperature  should  be  taken  nine  hours 
after  the  injection,  and  temperature  measurements 
repeated  at  regular  intervals  of  two  or  three  hours 
until  the  sixteenth  hour  after  the  injection. 

"  (d)  When  there  is  no  elevation  of  temperature  at 
this  time  (sixteen  hours  after  the  injection)  the  exam- 
ination may  be  discontinued;  but  if  the  temperature 
shows  an  upward  tendency,  measurements  must  be  con- 
tinued until  a  distinct  reaction  is  recognized  or  until 
the  temperature  begins  to  fall. 

"  (e)  If  a  reaction  is  detected  prior  to  the  sixteenth 
hour,  the  measurements  of  temperature  should  be  con- 
tinued until  the  expiration  of  this  period. 

"  (/)  If  there  is  an  unusual  change  of  temperature 
of  the  stable,  or  a  sudden  change  in  the  weather,  this 
fact  should  be  recorded  on  the  report-blank. 

"  (g)  If  a  cow  is  in  a  febrile  condition  tuberculin 
should  not  be  used,  because  it  would  be  impossible  to 
determine  whether,  if  a  rise  of  temperature  occurred, 
it  was  due  to  the  tuberculin  or  to  some  transitory  illness. 


294  BACTERIOLOGY. 

"  (Ji)  Cows  should  not  be  tested  within  a  few  days 
before  or  after  calving,  for  experience  has  shown  that 
the  result  at  these  times  may  be  misleading. 

"  (i)  The  tuberculin  test  is  not  recommended  for 
calves  under  three  months  old. 

"  (j)  In  old,  emaciated  animals  and  in  re-tests  use 
twice  the  usual  dose  of  tuberculin,  for  these  animals 
are  less  sensitive. 

"  (k)  Condemned  cattle  must  be  removed  from  the 
herd  and  kept  away  from  those  that  are  healthy. 

"  (/)  In  making  post-mortems  the  carcasses  should 
be  thoroughly  inspected,  and  all  of  the  organs  should 
be  examined." 

Tuberculin  injections  are  also  made  in  man  to  reveal 
a  suspected  tuberculosis.  At  first  some  believed  that  the 
irritation  aroused  in  the  tuberculous  foci  by  the  tuber- 
culin sometimes  caused  a  dissemination  of  the  bacilli  and 
an  increase  in  the  disease.  When  carefully  used,  how- 
ever, in  suitable  cases  there  is  probably  no  danger.  A 
drawback  to  its  usefulness  is  that  it  does  not  reveal  at 
all  the  extent  of  the  disease,  nor  whether  the  tuber- 
culosis is  active  or  dormant.  It  is,  however,  of  great 
value  in  selected  cases,  both  surgical  and  medical, 
where  slight  tuberculosis  is  suspected,  and  yet  no  de- 
cision can  be  reached.  I  quote  here  Dr.  Trudeau  upon 
the  use  of  the  test. 

"In  the  absence  of  any  well-defined  rules  founded 
upon  the  experience  of  others  at  the  time  I  began  to 
use  the  test,  the  method  I  adopted  has  been  a  purely 
arbitrary  one,  and  I  make  no  claim  for  its  being  the 
best  or  the  most  reliable,  although,  as  far  as  my  own 
personal  experience  goes,  I  have  as  yet  seen  no  objec- 
tion to  it  or  any  reason  to  modify  it. 


BACILLUS  OF  TUBERCULOSIS.  295 

"  The  range  of  the  patient's  temperature  is  ascer- 
tained by  taking  it  at  8  A.M.,  3  P.M.,  and  8  P.M.  for 
three  or  four  days  before  making  the  test.  The  first 
injection  should  not  exceed  0.5  mg.,  and  if  any  fever 
is  habitually  present  should  be  even  less,  and  is  best 
given  early  in  the  morning  or  late  at  night,  as  the 
typical  reaction  usually  begins,  in  my  experience, 
within  six  or  twelve  hours.  Such  a  small  dose,  while 
it  will  often  be  sufficient  to  produce  the  looked-for  rise 
of  temperature,  has,  under  my  observation,  never  pro- 
duced unpleasant  or  violent  symptoms.  An  interval  of 
two  or  three  days  should  be  allowed  between  each  of  the 
two  or  three  subsequent  injections  it  may  be  necessary 
to  give,  as  reaction  in  very  rare  cases  may  be  delayed 
for  twenty-four  or  even  thirty-six  hours.  On  the  third 
day  a  second  dose  of  1  mg.  is  given,  and  if  no  effect  is 
produced  a  third,  of  2  mg.,  three  days  later.  In  the 
great  majority  of  cases  of  latent  tuberculosis  an  appre- 
ciable reaction  will  be  produced  by  the  time  a  dose  of 
2  mg.  has  been  reached.  If  no  effect  has  been  caused 
by  the  tests  applied  as  above  I  have  usually  gone  no 
further,  and  concluded  that  no  tuberculous  process  was 
present,  or  at  least  not  to  a  degree  which  need  be  taken 
into  account  in  advising  the  patient  or  which  would 
warrant  insisting  on  a  radical  change  in  his  surround- 
ings and  mode  of  life.  If  some  slight  symptoms,  how- 
ever, have  been  produced  by  a  dose  of  2  mg.,  it  may 
be  necessary  to  give  a  fourth  injection  of  3  mg.  in  order 
to  reach  a  positive  conclusion.  Nevertheless,  it  should 
be  borne  in  mind  that  in  a  few  cases  the  exhibition 
of  even  larger  doses  may  cause  reaction  and  indicate 
the  existence  of  some  slight  latent  tuberculous  lesion, 
and  the  test  should  not,  when  applied  within  the 


296  BACTERIOLOGY. 

moderate    doses    described,  be    considered   absolutely 
infallible. 

"  No  evidence  in  connection  with  the  tuberculin  test 
as  applied  to  man  and  animals  has  been  forthcoming 
thus  far  from  those  who  have  made  use  of  it,  which 
would  tend  to  sustain  the  general  impression  that  this 
method  is  necessarily  dangerous  and  tends  invariably 
to  aggravate  the  disease,  and  my  own  experience  has 
developed  nothing  which  would  seem  to  confirm  this 
impression.  It  is  evident  that  the  size  of  the  doses 
given  has  much  to  do  with  the  limitations  of  this  method 
for  usefulness  and  the  correctness  of  the  conclusions 
reached  by  its  application.  The  tuberculin  used  is  also 
a  matter  of  some  importance  in  determining  the  dosage, 
as  different  samples  vary  considerably  in  their  efficiency. 
The  minute  amounts  adopted  by  Grasset  and  Vedel — 
i.  e.,  from  0.0002  to  0.0005— while  they  have  the  advan- 
tage of  absolute  safety,  may  lead  into  error,  as  they 
are  insufficient,  on  the  evidence  of  these  observers  them- 
selves, to  cause  reaction  in  cases  proven  to  be  tuberculous 
by  the  presence  of  the  bacillus  in  the  expectoration.  If, 
on  the  other  hand,  the  test  be  pushed  to  the  injection  of 
such  large  amounts  as  10  mg.  or  more,  as  advocated  by 
Maragliano,  such  doses  are  by  no  means  free  from  the 
objection  of  occasionally  causing  unpleasant  and  some- 
times dangerous  symptoms;  and  even  if  the  amount 
given  be  not  carried  to  the  dose  of  10  mg.,  which  is 
known  to  produce  fever  in  healthy  subjects,  it  is  likely 
that  on  account  of  individual  susceptibility  or  the  pres- 
ence of  some  other  morbid  process  in  the  body,  reaction 
will  be  found  to  occur  with  the  larger  doses  when  no 
tuberculous  process  exists.  The  adoption  of  an  initial 
dose  so  small  as  to  guard  against  the  absolute  possibility 


BACILLUS  OF  TUBERCULOSIS.  297 

of  producing  violent  reactionary  symptoms,  and  the 
graded  increase  of  the  subsequent  doses  within  such 
quantities  as  are  known  never  to  produce  reaction  in 
healthy  individuals,  would  seem  to  afford  the  best 
protection  against  unpleasant  results  and  misleading 
evidence. " 

Antituberculous  Serum.  Whether  serum-therapy  is 
destined  to  solve  the  problem  of  the  treatment  of  tuber- 
culosis remains  for  the  future  to  decide,  but  up  to  the 
present  the  results  obtained  with  antituberculous  serum 
do  not  warrant  our  forming  such  an  opinion.  The  at- 
tempts to  obtain  from  animals — chiefly  horses — a  serum 
which  would  be  protective  have  been  carried  out  along 
very  much  the  same  lines  as  Koch's  experiments  upon 
man.  The  methods  adopted  have  been  as  follows:  Old 
cultures  of  tubercle  bacilli  grown  in  5  per  cent,  glycerin 
bouillon  have  been  filtered  either  with  or  without  pre- 
vious boiling,  and  then  injected  into  animals,  this 
process  being  similar  to  Koch's  with  his  first  tuberculin. 
Others  have  injected  living  virulent  or  non-virulent 
tubercle  bacilli,  either  alone  or  with  their  culture  fluids; 
others  still  (Biichner)  have  injected  the  bacterial  proto- 
plasm obtained  by  crushing  tubercle  bacilli  together 
with  sand  and  squeezing  them  ;  this,  like  Koch  with 
his  new  tuberculin,  being  an  attempt  to  get  from  the 
unaltered  products  and  cell-contents  of  the  bacilli  the 
formation  in  the  body  of  bactericidal  or  immunizing 
substances. 

Among  the  many  claiming  good  results  in  man  or 
animals  thus  treated  may  be  mentioned  Hericourt, 
Bichet,  Bernheim,  Maragliano,  Yiquerat,  Paquin, 
de  Schweinitz  and  Dorset,  McFarland,  and  others. 
The  majority  claim  for  their  serum  the  power  to  neu- 


298  BACTERIOLOGY. 

tralize  the  effect  of  tuberculin  when  injected  into  tuber- 
culous guinea-pigs;  but  this  test  is  insufficient  and  prob- 
ably valueless,  since  tuberculin  is  not  the  same  as  the 
unaltered  products  of  the  tubercle  bacillus.  Moreover, 
it  has  been  shown  by  Trudeau  and  Baldwin  that  other 
substances  which  have  no  specific  properties  whatever 
will  have  much  the  same  effect  as  the  serum  under 
certain  conditions.  Some  make  the  further  claim  that 
guinea-pigs  injected  with  serum  acquire  an  immunity 
to  the  virulent  tubercle  bacilli,  and  that  those  already 
infected  live  longer  than  the  controls  which  receive  no 
serum;  and  some  even  claim  to  be  able  to  cure  animals 
eighteen  days  after  inoculation  with,  a  culture  of  tu- 
bercle bacilli.  Very  few  observers,  however,  have  suc- 
ceeded in  obtaining  appreciable  results  with  the  serums 
prepared  by  other  experimenters.  In  spite  of  such  con- 
flicting testimony,  it  is  probably  safe  to  assert  that  no 
serums  now  obtainable  have  any  great  value.  Nor  as 
we  look  at  the  progressive  nature  of  tuberculosis  can 
we  see  much  ground  to  hope  for  the  abundant  de- 
velopment of  curative  substances  in  the  blood  of  ani- 
mals. 

Prophylaxis.  Meanwhile  all  energies  should  be 
directed  to  the  prevention  of  tuberculosis,  not  only 
by  the  enforcement  of  proper  sanitary  regulations  as 
regards  the  care  of  sputum,  milk,  meat,  disinfection, 
etc.,  but  also  by  continued  experimental  work  and  by 
the  establishment  of  free  consumptive  hospitals,  and 
by  efforts  to  improve  the  character  of  the  food,  dwell- 
ings, and  condition  of  the  people  in  general,  we  should 
endeavor  to  build  up  the  individual  resistance  to  the 
disease.  It  may  be  years  yet  before  the  public  are 
sufficiently  educated  to  co-operate  with  the  sanitary 


BACILLUS  OF  TUBERCULOSIS.  299 

authorities  in  adopting  the  necessary  hygienic  measures 
to  stamp  out  tuberculosis  entirely;  but,  judging  from 
the  results  which  have  already  been  obtained  in  reducing 
the  mortality  from  this  dread  disease,  we  have  reason  to 
believe  that  in  time  it  can  be  completely  controlled. 

The  Tubercle  Bacillus  of  Cattle  and  its  Relation  to 
Human  Tuberculosis.  Among  the  domestic  animals 
tuberculosis  is  most  common  in  cattle.  On  account 
of  the  milk  which  they  provide  for  our  use,  and  which 
is  liable  to  contain  bacilli,  the  relation  of  these  to  human 
tuberculosis  is  a  matter  of  extreme  importance. 

The  chief  seat  of  the  lesions  is  apt  to  be  the  lungs 
and  with  them  the  pleura;  less  often  the  abdominal 
organs  and  the  udder  are  affected.  In  pigs  and  horses 
the  abdominal  organs  are  most  often  involved,  then  the 
lungs  and  lymphatic  glands.  In  sheep  and  goats  tuber- 
culosis is  rare.  The  bovine  bacillus,  as  the  most  im- 
portant of  the  group,  will  be  alone  considered  here. 

The  bacilli  derived  from  cattle  are  on  the  average  a 
little  shorter  and  straighter  than  the  average  human 
bacillus;  but  there  are  many  derived  from  cattle  exactly 
similar  to  those  derived  from  man  in  size,  shape,  and 
staining.  In  guinea-pigs,  and  especially  in  rabbits,  the 
bovine  bacilli  are  more  virulent  than  those  from  human 
sources.  Animals  infected  with  the  bacilli  from  cattle, 
as  well  as  those  from  the  other  domestic  animals,  react 
to  the  tuberculin  test.  All  these  bacilli  are,  therefore, 
undoubtedly  from  the  same  original  stock,  and  at 
first  glance  we  might  consider  it  unnecessary  to  prove 
that  those  derived  from  cattle  were  capable  of  causing 
human  tuberculosis.  There  are  facts,  however,  which 
tend  to  make  us  doubtful  of  the  extent  to  which  this 
infection  takes  place.  As  we  investigate  we  find  that 


300  BACTERIOLOGY. 

all  facts  tend  to  show  that  the  great  majority  of  cases, 
in  adults  at  least,  come  from  human  infection.  The 
cases  where  fairly  strong  proof  of  bovine  infection  has 
been  obtained  are  certainly  rare. 

Further,  we  have  the  undoubted  fact  that  constant 
sojourn  in  one  species  of  animal  tends  to  increase  the 
virulence  of  the  germ  for  that  animal  and  to  lessen  it 
for  others. 

Theobald  Smith  has  made  the  interesting  discovery 
that  there  is  a  wide  difference  between  the  culture 
growth  of  the  average  bovine  bacillus  and  the  average 
one  from  human  sources,  the  bovine  bacilli  being  shorter 
and  straighter,  and  growing  less  luxuriently  than  those 
from  man  ;  and,  further,  that  the  bovine  bacilli  are 
much  more  virulent  for  rabbits.  He  has  found  these 
differences  persist  for  long  periods,  and  believes  that 
the  simple  passage  through  a  single  person  in  a  case  of 
human  tnberculosis  would  not  be  sufficient  to  change 
these  characteristics.  He  has  not  yet  had  a  chance  to 
examine  the  bacilli  of  any  case  in  young  children  where 
milk  infection  was  strongly  suspected,  but  in  adults 
not  one  of  some  half  a  dozen  cultures  showed  the 
bovine  characteristics. 

At  present  it  seems  fair  to  assume  that  bovine  bacilli 
are  capable  of  infecting  only  those  that  are  very  sus- 
ceptible, such  as  young  children.  This  question  is  in 
great  need  of  further  study,  and  unless  proof  is 
brought  to  show  that  bovine  bacilli  never  infect  human 
beings,  no  cattle  which  are  shown  to  be  tubercular 
should  be  allowed  to  furnish  milk,  or  at  least  none  un- 
sterilized  should  be  used  for  drinking  purposes.  The  flesh 
is  less  harmful,  as  muscular  tissue  is  seldom  infected. 

Bird  (Avian)  Tuberculosis.    Tuberculosis  is  very  com- 


BACILLUS  OF  TUBERCULOSIS.  301 

mon  and  infectious  among  fowls.  The  bacilli  them- 
selves grow  more  readily  on  artificial  culture  media 
and  produce  a  more  even  and  moist  growth.  The 
bacilli  are  more  apt  to  show  branching  forms  than  the 
human.  In  rabbits  they  produce  very  similar  lesions. 
They  are  probably  from  the  same  stock  as  the  mamma- 
lian varieties;  but  it  is  not  believed  that  they  are  any, 
and  certainly  not  any  great,  factor  in  the  production  of 
human  tuberculosis. 

Diagnosis.  One  of  the  most  important  results  of  the 
discovery  of  the  tubercle  bacillus  relates  to  the  practical 
diagnosis  of  tuberculosis.  The  staining  peculiarities 
of  this  bacillus  render  it  possible  by  the  bacteriological 
examination  of  microscopical  preparations  to  make  an 
almost  absolutely  positive  diagnosis  in  the  majority  of 
cases.  A  still  more  certain  test  in  doubtful  cases  is 
the  subcutaneous  or  intraperitoneal  injection  of  guinea- 
pigs,  which  permits  of  the  determination  of  the  presence 
of  numbers  of  bacilli  so  small  as  to  escape  detection  by 
microscopical  examination.  For  the  animal  test,  how- 
ever, time  is  required — at  least  three  weeks,  and,  when 
the  result  is  negative,  several  months — before  any  posi- 
tive conclusion  can  be  reached,  for  when  only  a  few  ba- 
cilli are  present  tuberculosis  develops  slowly  in  animals. 

LABORATORY  TECHNIQUE  IN  THE  EXAMINATION 
FOR  TUBERCLE  BACILLI  AND  OTHER  ASSO- 
CIATED BACTERIA. 

I.   Microscopical  Examination  of  Sputum  for  the  Presence 
of  Tubercle  Bacilli. 

1.  Collection  of  Material.  The  sputum  should  be  col- 
lected in  a  clean  bottle  (two-ounce)  with  a  wide  mouth 
and  a  water-tight  stopper,  and  the  bottle  labelled  with 


302  BACTERIOLOGY. 

the  name  of  the  patient  or  other  distinguishing  mark. 
The  expectoration  discharged  in  the  morning  is  to  be 
preferred,  especially  in  recent  cases,  and  the  material 
should  be  coughed  up  from  the  lungs.  Care  should  be 
taken  that  the  contents  of  the  stomach,  nasopharyngeal 
mucus,  etc.,  are  not  discharged  during  the  act  of  expec- 
toration and  collected  instead  of  pulmonary  sputum.  If 
the  expectoration  be  scanty  the  entire  amount  discharged 
in  twenty-four  hoars  should  be  collected.  In  pul- 
monary tuberculosis  the  purulent,  cheesy,  and  muco- 
purulent  sputum  usually  contains  bacilli;  while  pure 
mucus,  blood,  and  saliva,  as  a  rule,  do  not.  When 
hemorrhage  has  occurred,  if  possible  some  purulent, 
cheesy,  or  mucopurulent  sputum  should  be  collected 
for  examination.  The  sputum  should  not  be  kept  any 
longer  than  necessary  before  examination,  for,  though 
a  slight  delay  or  even  till  putrefaction  begins,  does  not 
entirely  vitiate  the  result,  it  is  best  to  examine  it  in  as 
fresh  a  condition  as  possible. 

2.  Methods  of  Examination,  (a)  EXAMINATION  FOR 
TUBERCLE  BACILLI.  Pour  the  specimen  into  a  clean, 
shallow  vessel  having  a  blackened  bottom — a  Petr 
dish  placed  upon  a  sheet  of  dull  black  paper  answers 
the  purpose — and  select  from  the  sputum  one  of  the 
small,  white  or  yellowish-white,  cheesy  masses  or 
11  balls  "  which  it  is  seen  to  contain.  From  this  make 
a  cover-glass  "  smear "  in  the  usual  way.  Immerse 
this  in  a  solution  of  Ehrlich's  aniline-water  fuchsin  (see 
page  198),  contained  in  a  thin  watch-glass  or  porcelain 
dish,  and  steam  over  a  small  flame  for  two  minutes. 
Then  remove  the  cover-glass  from  this  and  wash  with 
water.  Now  decolorize  by  immersing  the  stained  prep- 
aration in  a  3  per  cent,  hydrochloric  acid  solution  in 


BACILLUS  OF  TUBERCULOSIS.  303 

alcohol  for  from  a  few  seconds  up  to  one  minute,  re- 
moving at  the  time  when  all  color  is  just  about  gone 
from  the  cover-glass  smear.  Wash  thoroughly  with 
water  and  make  a  contrast  stain  by  applying  a  cold 
solution  of  Loffler's  alkaline  methylene-blue — 

Concentrated  alcoholic  solution  of  methyl  blue     30  c.  c. 
Caustic  potash  (1:1 0,000  solution)         .         .100" 

for  from  fifteen  to  thirty  seconds.  Wash  with  water; 
press  between  folds  of  filter-paper;  dry  in  the  air; 
mount  and  examine. 

The  tubercle  bacilli  are  distinguished  by  the  fact  that 
they  retain  the  red  color  imparted  to  them  in  the 
fuchsin  solution,  while  the  other  bacteria  present, 
having  been  decolorized  in  the  acid  solution,  take  the 
contrast  stain  and  appear  blue.  (See  plate  II.,  Figs. 
1  and  2.) 

Various  methods  have  been  suggested  for  the  staining 
of  tubercle  bacilli,  but  the  original  method  as  employed 
by  Koch,  or  some  slight  modification  of  it,  is  so  satis- 
factory in  its  results  that  it  seems  unnecessary  to  substi- 
tute others  for  it.  The  above  is  a  slight  modification  of 
the  Koch-Ehrlich  method,  differing  from  it  chiefly  in 
the  use  of  a  weak  for  a  strong  acid  decolorizer.  It 
has  been  found  that  the  strong  acid  solution  originally 
employed  (5  per  cent,  sulphuric  acid  solution  in  alcohol) 
often  decolorizes  some  of  the  bacilli  entirely  by  its  too 
energetic  action,  and  that  a  weaker  decolorizer,  such 
as  the  above,  gives  more  uniform  results. 

Instead  of  the  Koch-Ehrlich  aniline-water  solution, 
ZiehVs  carbol-fuchsin  solution  may  be  used,  and  is  by 
many  preferred  (see  page  198).  Instead  of  floating 
the  cover-glass  smears  on  the  staining  fluid  they  can  be 


304  BACTERIOLOGY. 

held  in  the  Cornet  forceps,  covered  and  kept  covered 
completely  with  fluid  while  steamed  for  two  minutes 
over  the  flame. 

The  Koch-Ehrlich  solution  decomposes  after  having 
been  made  for  a  time,  so  that  it  must  be  freshly  pre- 
pared as  needed.  Solutions  older  than  fourteen  days 
should  not  be  used.  The  advantages  in  using  ZiehPs 
carbol-fuchsin  solution  are  that  it  keeps  well  and  is 
more  convenient  for  use  in  small  quantities. 

Another  method,  which  is  often  of  value  on  account 
of  its  simplicity  and  rapidity  of  performance,  is  that 
of  Frankel  as  modified  by  Gabbett.  This  consists  in 
staining  the  cover-glass  "  smear  "  with  steaming  ZiehPs 
carbol-fuchsin  solution  for  from  one  to  two  minutes, 
and  then  after  washing  in  water  placing  it  from  one- 
half  to  one  minute  directly  in  a  second  solution  which 
contains  both  the  acid  for  decolorizing  and  the  contrast 
stain.  This  second  solution  consists  of — 

Sulphuric  acid      .         .         .         .         .25  c.c. 
Methylene-blue  in  substance         .         .       2  grammes. 
Water          .         .        .        .         .         .     75  c.c. 

It  is  then  washed  with  water  and  is  ready  for  examina- 
tion. The  tubercle  bacilli  will  remain  red  as  stained  by 
the  fuchsin,  while  all  other  bacteria  will  be  tinted  blue. 
When  the  number  of  tubercle  bacilli  in  sputum  is 
very  small  they  may  easily  escape  detection.  Methods 
have,  therefore,  been  suggested  for  finding  them  under 
these  circumstances.  Ribbert  proposed  the  addition  to 
the  sputum  of  a  2  per  cent,  solution  of  caustic  potash 
and  boiling  the  mixture.  The  mucus  is  dissolved,  and 
when  the  mixture  is  placed  in  a  conical  glass  vessel  any 
bacilli  present  are  deposited  at  the  bottom,  and  may  be 
found  in  the  sediment  after  removing  the  supernatant 


BACILLUS  OF  TUBERCULOSIS.  3Q5 

fluid.    The  sedimentation  may  be  obtained  more  quickly 
by  the  centrifugal  machine. 

II.  Examination  for  Other  Bacteria  (Mixed  Infection). 

With  regard  to  the  bacteriological  diagnosis  of  pul- 
monary phthisis,  many  consider  that  it  is  not  enough 
to  show  only  the  presence  of  tubercle  bacilli;  it  is  held 
to  be  of  equal  importance,  both  for  purposes  of  prognosis 
and  treatment,  that  the  presence  of  other  micro-organ- 
isms which  may  be  associated  with  the  tubercle  bacillus 
should  also  be  determined.  It  is  now  usual  to  dis- 
tinguish pure  tuberculosis  of  the  lungs  from  a  mixed 
infection.  Phthisis  due  to  the  tubercle  bacillus  alone, 
which  constitutes  but  a  small  percentage  of  all  cases, 
may  occur  without  febrile  reaction;  or  when  fever  occurs 
the  prognosis  is  unfavorable,  thus  indicating  that  the 
disease  is  already  advanced.  It  is  in  the  uncomplicated 
forms  of  phthisis,  moreover,  where  one  must  expect  if 
anywhere  the  best  results  from  treatment  with  tuber- 
culin or  antituberculous  serum.  The  majority  of  cases, 
however,  of  pulmonary  tuberculosis  show  a  mixed  in- 
fection, especially  with  varieties  of  the  streptococcus 
and  pneumococcus.  These  cases  may  be  active,  with 
fever,  or  passive,  without  fever,  according,  perhaps, 
as  the  parenchyma  of  the  lung  is  invaded  by  the  bac- 
teria; or  they  are  only  superficially  located  in  cavities, 
bronchi,  etc.  Mixed  infection  with  the  staphylococcus 
and  with  the  influenza  and  pneumonia  bacilli  have 
also  been  frequently  met  with  by  us.  The  tetragenus 
has  not  been  detected  by  us  in  thoroughly  washed 
fresh  sputum,  but  has  been  found  by  others.  At  present 
the  facts  seem  to  prove  that  the  tubercle  bacilli  have 
in  the  great  majority  of  cases  at  least  until  shortly 

20 


306  BACTERIOLOGY. 

before  death,  a  more  important  role  than  the  associated 
bacteria. 

The  great  majority  of  stained  smears  from  specimens 
of  sputa  show  not  only  the  tubercle  bacilli  stained  in 
red,  but  many  other  bacteria  stained  blue.  Some  of 
these  associated  bacteria  have  come  from  the  diseased 
areas  of  the  lungs,  while  others  were  merely  added 
to  the  sputa  as  it  passed  through  the  mouth,  or  have 
developed  after  gathering.  To  separate  the  one  from 
the  other  we  wash  the  sputa. 

Sputum  Washing.  The  first  essential  is  that  the 
material  be  washed  within  a  few  minutes,  and  certainly 
within  an  hour,  of  being  expectorated.  If  a  longer 
time  is  allowed  to  intervene,  the  bacteria  from  the 
mouth  will  penetrate  into  the  interior  of  the  mucus,  and 
thus  appear  as  if  they  came  from  the  lungs.  Sputum 
treated  twenty-four  hours  after  its  expectoration  is  use- 
less for  examining  for  anything  except  the  tubercle 
bacillus.  A  rough  method  is  to  pour  some  of  the  speci- 
men of  sputum  to  be  examined  into  a  convenient  re- 
ceptacle containing  sterile  water,  and  withdraw,  by 
means  of  a  sterilized  platinum  wire,  one  of  the  cheesy 
masses  or  thick  "  balls'7  of  mucus.  Pass  this  loop  five 
times  through  sterile  water  in  a  dish;  repeat  the  oper- 
ation in  freshwater  in  a  second  and  third  dish.  Spread 
what  remains  of  the  mass  on  cover -glasses  and  make 
smear  preparations;  stain  and  examine.  With  another 
mass  inoculate  ascitic  bouillon  in  tubes  and  agar  in 
plates. 

If  it  is  desired  to  examine  the  specimens  for  capsule 
bacteria,  pneumococci,  etc.,  they  may  be  stained  by 
Welch's  acetic-acid  method  (page  203)  or  by  Gram's 
method  (page  203). 


BACILLUS  OF  TUBERCULOSIS.  307 

When  we  wish  to  thoroughly  exclude  mouth  bacteria 
a  lump  of  the  sputum  raised  by  a  natural  cough  is  seized 
by  the  forceps  and  transferred  to  a  bottle  of  sterile 
water  and  thoroughly  shaken ;  it  is  then  removed  to  a 
second  bottle  of  bouillon  and  again  thoroughly  shaken. 
From  this  it  is  passed  in  the  same  way  through  four 
other  bottles  of  bouillon.  A  portion  of  the  mass  is 
now  smeared  over  cover-glasses,  and  the  rest  inoculated 
in  suitable  media,  such  as  agar  in  Petrie  dishes,  and 
ascitic  fluid  bouillon  in  tubes.  If  desired  the  bacteria 
washed  .  off  in  the  different  washings  are  allowed  to 
develop. 

Practical  Notes  on  the  Examination  for  Mixed  Infec- 
tion. 1.  The  difficulties  to  be  overcome,  in  order  to 
obtain  sputum  consisting  presumably  of  exudate  from 
the  deeper  portions  of  the  lungs,  are  so  great  that  the 
collection  of  the  specimens  should  be  supervised  by  the 
bacteriologist  in  charge  of  the  work  of  examination. 

2.  Specimens   of    sputum  collected  even  with    the 
greatest   precaution   may   give    evidence   of     decided 
mouth  infection  unless  immediately  washed. 

3.  The  sputum  must  be  examined  very  soon  after 
collection. 

4.  The  culture  medium  used  for  the  final  cultures 
must  be  suitable  for  the  growth  of  the  micro-organisms. 

5.  At  least  two  successive  examinations  of  sputum 
should  be  made  in  each  case. 

6.  The  results,  especially  as  to  the  number  of  colo- 
nies, vary  according  to  the  size  and  tenacity  of  the  ball 
of  sputum  washed — e.g.,  a  small  ball  of  sputum  which 
becomes  more  or  less  broken  up  upon  thorough  shaking 
may  contain  very  few  or  no  bacteria. 

Williams,  in  the  examination  of  the  sputum  in  some 


308  BACTERIOLOGY. 

forty  cases,  came  to  the  following  conclusions  :  1.  The 
presence  of  a  large  number  of  bacteria  in  a  satisfactory 
and  thoroughly  washed  specimen  of  sputum  indicates 
that  these  bacteria  probably  play  an  active  part  in  the 


2.  The  presence  of  a  small  number  of  bacteria  in 
such  sputum  does  not  necessarily  indicate  that  they  are 
not  active  in  that  case,  for  they  may  penetrate  more  or 
less  deeply  into  the  lung  tissue,  and  produce  patho- 
logical changes  without  being  thrown  off  in  large  num- 
bers with  the  exudate.     It  is  probable,  however,  that, 
as  a  rule,  the  smaller  the  number  found  the  less  the 
degree  of  mixed  infection. 

3.  Cases  of  clinically  secondary  infection  frequently 
give  pure  cultures  of  some  one  organism  which  appeared 
to  be  capable  of  causing  the  symptoms. 

4.  In  the   majority   of    severe   cases   of    clinically 
mixed  infection  many  organisms  have  been  found  which 
usually  have  belonged  to  several  different  species  or 
varieties. 

5.  In  the  majority  of  cases  of  clinically  non-mixed 
infection  very  few  organisms  have  been  found. 

6.  Only  bacteria   which   might   cause   pathological 
changes  were  present. 

7.  Very  few  of  the  organisms  found  were  virulent 
in  rabbits,  even  though   coming  from  severe  cases  of 
mixed  infection. 

The  virulence  for  laboratory  animals  of  bacteria  ob- 
tained from  the  sputum  is,  therefore,  no  indication  of 
their  virulence  for  man,  because  of  the  impossibility  of 
reproducing  in  such  animals  the  exact  condition  of  sus- 
ceptibility present  in  human  infection. 

General  Rules  in  Microscopical  Examination  of  Sputum. 


BACILLUS  OF  TUBERCULOSIS.  309 

Always  make  two  cover-glass  preparations  from  each 
specimen.  Report  no  result  as  negative  until  at  least 
two  preparations  have  been  subjected  to  a  thorough 
search  with  a  1/12  oil-immersion  or  2  mm.  apochromatic 
lens  by  means  of  a  mechanical  stage.  From  a  very 
large  experience  in  the  examination  of  sputum  for 
tubercle  bacilli,  the  New  York  Health  Department 
bacteriologists  have  concluded  that  the  examination 
of  two  preparations  of  each  specimen  in  the  careful 
manner  described  above  is  usually  sufficient  to  demon- 
strate the  presence  of  the  bacilli  when  they  are  pres- 
ent in  the  sputa,  and  they  are  usually  found  to  be 
present  to  this  extent  in  fairly  well-developed  cases 
of  pulmonary  tuberculosis,  and  in  many  cases  which 
are  in  the  incipient  stage.  There  are,  however,  un- 
doubted cases  of  incipient  pulmonary  tuberculosis  which 
require  the  examination  of  many  preparations  before 
the  tubercle  bacillus  can  be  found;  and  that  cases  also 
occur  in  which  the  sputum  for  a  time  does  not  contain  the 
bacilli,  which  were,  nevertheless,  present  at  an  earlier 
period,  and  which  again  later  appear.  Therefore,  if  cases 
occur  which  may  be  still  regarded  as  possibly  tubercu- 
losis, further  examinations  of  the  sputum  should  be 
made.  It  should  also  be  constantly  borne  in  mind 
that  the  demonstration  of  the  presence  of  tubercle 
bacilli  in  the  sputum  proves  about  as  conclusively  as 
anything  can  the  existence  of  some  degree  of  tubercu- 
losis; but  that  the  absence  of  tubercle  bacilli  or  the 
failure  to  find  them  microscopically  does  not  positively 
exclude  the  existence  of  the  disease.  Here  injections 
of  tuberculin  can  be  made  use  of. 


310  BACTERIOLOGY. 

III.   Staining  of  Tubercle  Bacilli  in  Tissues. 

Thin  sections  of  tuberculous  tissues  may  be  stained 
by  the  same  methods  recommended  for  cover-glass 
preparations,  except  that  it  is  best  not  to  employ  heat 
to  any  extent. 

The  Ehrlich's  Method.  Place  the  paraffin  sections  in 
aniline  fuchsin  and  leave  for  from  one  to  twelve  hours; 
then  decolorize  by  placing  them  for  about  half  a  minute 
in  dilute  nitric  acid  (10  per  cent.);  wash  in  60  per  cent, 
alcohol  until  no  more  color  is  given  off;  counter  stain 
for  two  or  three  minutes  in  a  saturated  aqueous  solu- 
tion of  methylene-blue,  wash  in  water;  dehydrate  with 
absolute  alcohol;  clear  up  in  oil  of  cedar  or  xylol  and 
mount  in  xylol  balsam. 

Method  of  Ziehl-Neelson.  Stain  the  section  in  warmed 
carbol-fuchsin  solution  for  one  hour,  the  temperature  to 
be  not  over  45°  to  50°  C.  Decolorize  for  a  fe\y  seconds 
in  5  per  cent,  sulphuric  acid,  then  in  70  per  cent,  alcohol, 
and  from  this  on  as  in  the  Ehrlich  method. 


CHAPTER  XIX. 

BACILLI  SHOWING  SIMILAR  STAINING  REACTIONS  TO 
THOSE  OF  THE  TUBERCLE  BACILLI  —  SYPHILIS 
BACILLUS — SMEGMA  BACILLUS — LEPROSY  BACIL- 
LUS— GRASS  BACILLI. 

SYPHILIS  BACILLUS. 

DISCOVERED  by  Lustgarten  in  syphilitic  lesions  and 
secretions  of  syphilitic  ulcers  (1884),  and  believed  by 
him  to  be  the  specific  .cause  of  this  disease.  It  has 
since  been  shown  that  in  normal  smegma  from  the 
prepuce  or  the  vulva  bacilli  are  found  in  great  abun- 
dance, similar  in  their  morphology  to  the  bacillus  of 
Lustgarten,  but  differing,  as  a  rule,  slightly  in  certain 
staining  peculiarities.  (See  Fig.  39,  page  313.) 

Morphology.  Straight  or  curved  bacilli,  which  bear 
considerable  resemblance  to  tubercle  bacilli,  but  differ 
from  them  in  staining  reactions.  They  are  from  3  to 
5//  long  and  from  0.2  to  0.3/*  broad,  usually  curved 
or  bent  at  a  sharp  angle,  or  S-shaped,  often  thickened  at 
one  end  and  irregularly  notched.  With  a  high-power 
lens  bright,  shining  spaces  in  the  deeply  stained  rods 
may  be  observed;  these,  from  two  to  four  in  a  single  rod, 
are  believed  by  Lrustgarten  to  be  spores.  The  bacilli 
are  not  usually  found  free  in  the  tissues,  but  commonly 
lie  singly  or  sometimes  in  groups  within  the  interior  of 
cells  having  a  round,  oval,  or  polygonal  form,  and 
apparently  somewhat  swollen. 


312  BACTERIOLOGY. 

The  bacillus  of  Lustgarten  stains  with  equal  diffi- 
culty as  the  tubercle  bacillus,  but  is  much  less  resistant 
to  the  action  of  certain  decolorizing  agents,  such  as 
mineral  acids,  particularly  sulphuric  acid.  It  is,  as  a 
rule,  more  resistant  to  the  decolorizing  action  of  alcohol 
than  the  smegma  bacillus. 

Biological  and  Pathogenic  Properties.  Numerous  at- 
tempts have  been  made  to  cultivate  the  bacillus  of 
Lustgarten  on  artificial  media,  but  without  success. 
The  inoculation  of  animals  with  syphilitic  tissues  and 
secretions  has  also  given  only  negative  results,  though 
in  man,  as  is  well  known,  such  inoculation  has  often 
taken  place,  the  tertiary  products  only  being  non- 
infectious;  but  as  the  bacillus  has  never  been  obtained 
in  pure  culture,  we  have  no  ppsitive  information  as  to 
its  biological  characters  or  pathogenesis. 

Lustgarten' s  bacillus  has  been  found  in  various  syph- 
ilitic tissues  and  lesions,  in  beginning  sclerosis,  in  the 
papules,  in  condylomata  and  gummata,  and  not  only 
in  the  vicinity  of  the  genitals,  but  also  in  the  mouth, 
throat,  heart,  and  brain.  No  satisfactory  experimental 
evidence  has  been  given  of  its  causative  relation  to 
syphilis,  but  the  failure  to  find  other  micro-organisms, 
and  the  occurrence  of  these  characteristic  bacilli  in  vari- 
ous parts  of  the  body,  would  seem  to  point  to  their  etio- 
logical  importance;  while,  on  the  other  hand,  the  long 
immunity  in  syphilis,  so  different  from  that  in  any 
known  bacterial  disease,  casts  doubt  not  only  on  the 
status  of  this  bacillus,  but  also  upon  the  bacterial 
nature  of  the  micro-organism.  The  fact  that  the  ba- 
cilli have  been  found  occasionally  in  tertiary  lesions — 
which,  however,  are  known  to  possess  no  infectious 
property — may  possibly  be  explained  by  the  some- 


SYPHILIS  BACILL US.  313 

what  improbable  assumption  that  the  bacilli  here 
present  have  become  attenuated  or  have  died.  The 
finding  of  saprophytic  bacilli — the  so-called  smegma 
bacilli — (Fig.  39  and  Plate  I.,  Fig.  4),  almost  identical 


FIG.  39. 


S 


Smegma  bacilli,  similar  in  appearance  to  syphilis  bacilli.  X  1000  diam. 

morphologically  with  the  bacillus  of  Lustgarten,  under 
the  prepuce  of  healthy  persons,  does  not  prove  the 
identity  of  the  two  bacilli,  though  in  the  absence  of 
cultures  and  inoculation  experiments  we  have  not  the 
means  of  establishing  their  relationship  to  one  another. 
The  smegma  bacilli  have  never  been  identified  in  other 
parts  of  the  body  except  in  the  neighborhood  of  the 
genitals.  While  the  bacillus  of  Lustgarten  cannot 
resist  the  prolonged  decolorizing  action  of  acids,  but 
is  resistant  to  the  action  of  alcohol,  the  smegma  ba- 
cillus, when  stained,  is  quickly  decolorized  by  alcohol, 
but  quite  resistant  to  5  per  cent,  sulphuric  acid  solution. 
Beside,  the  syphilis  bacillus  has  been  found  in  papules, 
in  gummata,  and  other  syphilomata  where  there  seems 
no  probability  whatever  of  the  smegma  bacillus  having 
emigrated.  Baumgarten,  who  has  searched  in  vain  for 
Lustgarten' s  bacillus  in  uncomplicated  visceral  syphilo- 


314  BACTERIOLOGY. 

mata,  suggests  that  the  bacilli  found  in  such  lesions  were, 
perhaps,  tubercle  bacilli,  and  represented  a  mixed  in- 
fection. This  may  have  been  true  of  some  cases,  no 
doubt,  as  the  differentiation  of  new-growths  of  tertiary 
syphilis  and  tuberculosis  is  often  difficult;  but  the  differ- 
entiation of  the  two  bacilli  can  usually  be  made  by  their 
different  powers  of  resistance  to  the  decolorizing  action 
of  acids.  Finally,  other  micro-organisms  have  been 
described  and  claimed  to  be  the  specific  cause  of  syph- 
ilis, but  none  of  these  discoveries  have  been  confirmed. 
From  this  it  appears  that  though  there  is  no  conclusive 
proof  of  the  fact,  there  is  some  possibility,  but  hardly 
a  probability,  that  Lustgarten's  bacillus  is  the  true  cause 
of  syphilis.  Its  position  at  present  is  too  doubtful  to 
make  its  detection  of  any  diagnostic  value. 

Syphilitic  Infection.  Infection  of  those  not  immune 
can  take  place  at  any  time  when  an  abrasion,  however 
small,  is  brought  in  contact  with  the  blood  or  secretions 
from  the  primary  or  secondary  lesions  of  syphilitics. 

The  differential  diagnosis  of  Lustgarten's  bacillus 
must  be  made  from  the  tubercle  bacillus,  the  smegma 
bacillus,  and  the  leprosy  bacillus.  According  to  Hueppe, 
the  differential  diagnosis  between  these  four  organisms 
depends  upon  the  following  reactions  :  When  stained 
by  the  carbol-fuchsin  method  commonly  employed  in 
staining  the  tubercle  bacillus,  the  syphilis  bacillus  be- 
comes almost  instantly  decolorized  by  treatment  with 
mineral  acids,  particularly  sulphuric  acid;  whereas  the 
smegma  bacillus  resists  such  treatment  for  a  much 
longer  time,  and  the  lepra  and  tubercle  bacillus  for  a 
still  longer  time.  On  the  other  hand,  if  decolorization 
is  practised  with  alcohol  instead  of  acids  the  smegma 
bacillus  is  the  first  to  lose  its  color.  The  bacillus  tuber- 


LEPROSY  BA  GILL  US.  315 

culosis  and  the  bacillus  of  leprosy  are  both  very  retentive 
of  their  color,  even  after  treatment  with  acids  and 
alcohol.  If,  then,  we  treat  the  preparation,  stained  with 
carbolfuchsin,  with  sulphuric  acid,  the  syphilis  bacillus 
becomes  almost  at  once  decolorized.  If  it  is  not  imme- 
diately decolorized,  treat  with  alcohol;  if  it  is  then 
decolorized,  it  is  the  smegma  bacillus.  If  it  is  still 
not  decolorized,  it  is  either  the  leprosy  or  the  tubercle 
bacillus. 

By  these  methods  the  differential  diagnosis  can  usually 
be  made.  In  all  investigations  of  importance,  however, 
animal  inoculations  should  also  be  made,  as  by  this 
means  alone  can  a  positive  diagnosis  from  tuberculosis 
be  established.  Especial  care  should  be  observed  in 
the  examination  of  syphilitic  ulcers  of  the  genital  re- 
gion, as  in  this  situation  the  smegma  bacilli  are  almost 
always  present. 

LEPROSY  BACILLUS. 

The  bacillus  of  leprosy  was  discovered  by  Hansen 
and  Neisser  (1879)  in  the  leprous  tubercles  of  persons 
afflicted  with  the  disease.  This  discovery  was  confirmed 
by  many  subsequent  observers. 

Morphology.  Small,  slender  rods  resembling  the 
tubercle  bacilli  in  form,  but  somewhat  shorter  and 
not  so  frequently  curved.  The  rods  have  pointed  ends, 
and  in  stained  preparations  unstained  spaces,  similar  to 
those  observed  in  the  tubercle  bacillus,  are  seen.  They 
stain  readily  with  the  aniline  colors  and  also  by  Gram's 
method.  Although  differing  from  the  tubercle  bacillus 
in  the  ease  with  which  they  take  up  the  ordinary  aniline 
dyes,  they  behave  like  tubercle  bacilli  in  retaining  their 


316  BACTERIOLOGY. 

color  when  subsequently  treated  with  strong  solutions 
of  the  mineral  acids  and  alcohol.  Thus  double-stained 
preparations  may  be  made  by  first  staining  sections  or 
cover-glass  preparations  in  ZiehPs  carbol-fuchsin  solu- 
tion or  in  an  aqueous  solution  of  methyl-violet,  de- 
colorizing in  acid,  washing  in  alcohol,  and  counter- 
staining  with  methylene-blue  or  fuchsin. 

Biological  Characters.  Attempts  to  cultivate  the  bacil- 
lus leprse  have  been  frequently  made,  but  so  far  with 
only  questionable  results.  None  of  the  cultures  ob- 
tained have  given  positive  results  when  inoculated 
into  animals. 

Pathogenesis.  Numerous  inoculation  experiments 
have  been  made  on  animals  with  portions  of  leprous 
tubercles,  excised  for  the  purpose  from  lepers,  but 
although  a  few  positive  results  have  been  reported, 
there  is  no  conclusive  evidence  that  leprosy  can  be 
transmitted  to  the  lower  animals  by  inoculation.  The 
inference  that  this  bacillus  bears  an  etiological  relation 
to  the  disease  with  which  it  is  associated  is  based 
entirely  upon  the  demonstration  of  its  constant  pres- 
ence in  leprous  tissues. 

The  bacilli  are  found  in  all  the  diseased  parts  and 
usually  in  large  numbers,  especially  in  tubercles  on  the 
skin,  in  the  conjunctiva  and  cornea,  and  the  mucous 
membranes  of  the  mouth,  gums,  and  larynx,  and  in 
the  interstitial  processes  of  the  nerves,  the  testicles, 
spleen,  liver,  and  kidneys.  The  rods  lie  almost  exclu- 
sively within  the  peculiar  round  or  oval  cells  of  the 
granulation  tissue  which  composes  the  leprous  tubercles, 
either  irregularly  scattered  or  arranged  parallel  to  one 
another.  In  old  centres  of  infection  the  leprosy  cells 
containing  the  bacilli  are  larger  and  often  polynuclear. 


LEPROSY  BACILLUS.  317 

Giant-cells,  such  as  are  found  in  tuberculosis,  are  claimed 
to  have  been  observed  by  a  few  investigators  (Boinet 
and  Borrel).  In  the  interior  of  the  skin  tubercles  the 
hair  follicles,  sebaceous  and  sweat-glands  are  often 
attacked,  and  bacilli  have  sometimes  been  found  in 
these  (Unna,  etc.).  Quite  young  eruptions  often  con- 
tain a  few  bacilli.  A  true  caseation  of  the  tubercles 
does  not  occur,  but  ulceration  results. 

In  the  anaesthetic  forms  of  leprosy  the  bacilli  are 
found  most  commonly  in  the  nerves  and  less  frequently 
in  the  skin.  They  have  been  demonstrated  in  the  sym- 
pathetic nervous  system,  in  the  spinal  cord,  and  in  the 
brain.  The  bacillus  leprae  occurs  also  in  the  blood, 
partly  free  and  partly  within  the  leucocytes,  especially 
during  the  febrile  stage  which  precedes  the  breaking 
out  of  fresh  tubercles  (Walters  and  Doutrelepont).  The 
bacilli  have  also  been  found  in  the  intestines,  in  the 
lungs,  and  in  the  sputum,  but  not  in  the  urine. 

With  regard  to  the  question  of  the  direct  inheri- 
tance of  the  disease  from  the  mother  to  the  unborn 
child  there  is  considerable  difference  of  opinion.  Some 
cases  have  been  reported,  however,  in  which  a  direct 
transmission  of  the  bacillus  during  intra-uterine  life 
seems  to  have  been  the  only  or  most  plausible  expla- 
nation of  the  infection.  At  the  same  time,  we  have  no 
positive  experimental  evidence  to  prove  that  such  an 
infection  does  take  place.  Although  many  attempts 
have  been  made  to  infect  healthy  individuals  with 
material  containing  the  bacilli  of  leprosy,  the  results 
are  not  conclusive.  Even  the  experiments  made  by 
Arning,  who  inoculated  a  condemned  criminal  in  the 
Sandwich  Islands  with  fresh  leprous  tubercles,  and 
which  has  been  generally  regarded  as  positive  evidence 


318  BACTERIOLOGY. 

of  the  transmissibility  of  the  disease  in  this  way,  is  by 
no  means  conclusive;  for,  according  to  Swift,  the  man 
had  other  opportunities  for  becoming  infected.  These 
negative  results,  together  with  the  fact  that  infection 
does  not  more  frequently  occur  in  persons  exposed  to 
the  disease,  may  possibly  be  explained  by  the  assump- 
tion that  the  bacilli  contained  in  the  tubercular  tissue 
are  mostly  dead,  or  much  more  probably  that  an  indi- 
vidual susceptibility  to  the  disease  is  requisite  for  its 
production. 

The  wide-spread  idea,  before  the  discovery  of  the 
leprosy  bacillus,  that  the  disease  was  associated  with  the 
constant  eating  of  dried  fish  or  a  certain  kind  of  food 
has  now  been  entirely  abandoned. 

The  relation  of  leprosy  to  tuberculosis  is  sufficiently 
evident  from  their  great  similarity  in  many  respects. 
This  is  rendered  still  more  remarkable  if  the  ob- 
servation recently  made  is  true,  that  leprosy  reacts, 
both  locally  and  generally,  to  an  injection  of  tuberculin 
in  the  same  manner  as  tuberculosis  (Babes  and  Kalin- 
dero). 

Differential  Diagnosis.  The  differential  diagnosis  be- 
tween leprosy  and  tuberculosis  is  not  difficult  in  typical 
cases.  The  large  numbers  of  bacilli  found  in  the  inte- 
rior of  the  cells  would  point  with  great  probability  to 
leprosy.  Too  much  importance  should  not  be  placed 
upon  the  staining  peculiarities,  as  these  are  not  con- 
stant. Moreover,  the  two  diseases  not  infrequently 
occur  together  in  the  same  individual.  In  making  the 
diagnosis,  therefore,  all  the  signs,  histological  and 
pathogenic,  must  be  considered  and  animal  inoculations 
made. 


TIMOTHY  AND  OTHER  GRASS  BACILLI.     319 

TIMOTHY  AND  OTHER  GRASS  BACILLI. 

On  various  grasses,  in  cow's  manure,  in  butter,  and  in 
milk,  there  have  been  discovered  a  number  of  varieties 
of  bacteria  which  have  more  or  less  of  the  characteristics 
of  the  tubercle  bacillus.  Some  of  them  are  as  difficult  to 
stain  and  as  resistant  to  decolorizing  action  of  mineral 
acids  and  alcohol  as  the  tubercle  bacillus  found  in  man. 
Many  of  them  are  of  the  same  general  size  and  shape  as 
the  tubercle  bacillus,,  and,  strangely  enough,  produce 
in  animals  small  diseased  areas,  which  not  only  macro- 
scopically  but  also  microscopically  resemble  miliary 
tubercles  due  to  the  tubercle  bacillus.  They,  however, 
are  entirely  different  in  their  culture  characteristics, 
producing  in  twenty-four  to  forty-eight  hours  on  ordi- 
nary culture  media  moist,  round  colonies  of  an  eighth 
to  a  quarter  of  an  inch  in  diameter,  and  of  a  more  or 
less  intense  pink  color.  In  animals  they  produce  only 
localized  lesions,  causing  death  only  when  injected  in 
large  numbers.  The  injected  animals  are  unaffected 
by  tuberculin  injections.  The  chief  interest  which 
these  bacilli  have  for  us  is  the  possibility  of  confusing 
them  with  the  tubercle  bacilli.  This  danger  is  always 
present  in  milk,  for  the  grass  bacilli  find  so  many 
means  of  gaining  entrance  to  it.  In  the  examination 
of  dust,  healthy  throat  and  nose  secretions,  etc.,  the 
simple  microscopical  examination  might  lead  to  error. 

They  can  be  separated  from  tubercle  bacilli  by  in- 
oculating animals,  and  then  if  they  show  any  infection, 
injecting  tuberculin,  when  if  infected  with  tuberculosis 
they  will  die,  but  if  by  grass  bacilli  they  will  show 
no  reaction.  Cultures  from  the  lesions  will  also  show 
on  ordinary  media  pink  colonies  if  grass  bacilli  are 
present,  and  no  growth  if  only  tubercle  bacilli. 


CHAPTEE   XX. 


INFLUENZA   BACILLUS. 


AFTER  numerous  unsuccessful  attempts  during  the 
epidemic  of  1889  and  succeeding  years  to  discover  the 
specific  cause  of  influenza,  Pfeiffer  succeeded  in  isolating 
a  bacillus  (1892)  from  the  purulent  bronchial  secretion 
of  patients  suffering  from  epidemic  influenza  which  he 


FIG.  40. 


Influenza  bacilli.    X  1100  diameters. 

showed  was  the  probable  cause  of  the  disease.  This 
discovery  has  since  been  confirmed  by  many  observers, 
the  results  of  whose  researches  give  us  reason  to  believe 
that  this  bacillus  is  the  chief  etiological  factor  in  the 
production  of  influenza. 


INFLUENZA  BACILLUS.  321 

Morphology.  Very  small,  moderately  thick  bacilli, 
usually  occurring  singly  or  united  in  pairs,  but  threads 
or  chains  of  three,  four,  or  more  elements,  are  occa- 
sionally found. 

The  bacillus  stains  with  difficulty  with  the  ordinary 
aniline  colors — best  with  dilute  ZiehPs  solution  or 
Loffler's  methylene-blue  solution,  with  heat.  When 
faintly  stained  the  two  ends  of  the  bacilli  are  some- 
times more  deeply  stained  than  the  middle  portion. 
Those  we  have  examined,  all  obtained  from  cases  in 
New  York,  were  not  stained  by  Gram's  method,  but 
some  report  instances  in  which  they  were. 

Biological  Characters.  An  aerobic,  non-motile  bacil- 
lus; does  not  form  spores;  no  growth  occurs  below 
26°  C.,  or  above  43°  C.,  or  in  the  entire  absence  of  oxy- 
gen. This  bacillus  is  best  cultivated  at  37°  C.,  and  on 
the  surface  of  the  ordinary  nutrient  culture  media  con- 
taining haemoglobin  or  purulent  material.  Plain  or 
glycerin-agar,  or  blood-serum  streaked  with  sputum, 
pus,  or  blood,  make  a  good  soil  for  their  growth.  At 
the  end  of  eighteen  to  twenty-four  hours  in  the  incu- 
bator very  small,  drop-like  colonies  are  developed, 
which,  under  a  low  magnification  (100  diameters),  ap- 
pear as  shining,  transparent,  homogeneous  masses,  and 
even  under  a  No.  7  lens  scarcely  show  at  all  the  indi- 
vidual organisms.  Older  colonies  are  sometimes  col- 
ored yellowish-brown  in  the  centre.  A  characteristic 
feature  of  the  influenza  bacillus  is  that  the  colonies  tend 
to  remain  separate  from  each  other,  although  when  they 
are  thickly  sown  in  a  film  of  moist  blood  upon  nutri- 
ent agar  they  may  become  confluent.  Transplantation 
of  the  original  culture  to  ordinary  agar  or  serum  can- 
not, as  a  rule,  be  successfully  performed,  owing  to  the 

21 


322  BACTERIOLOGY. 

want  of  sufficient  haemoglobin;  but  if  sterile  rabbit, 
pigeon,  or  human  blood  be  added  to  these  media  trans- 
plantation may  be  indefinitely  performed,  provided  it  is 
done  every  three  or  four  days.  Cultures  may  remain 
alive  up  to  seventeen  days  in  the  ice-chest. 

The  Detection  of  the  Influenza  Bacillus  in  Sputum. 
When  it  is  desired  to  obtain  cultures  of  the  bacillus 
of  influenza  for  diagnostic  purposes  from  material  sus- 
pected to  contain  this  organism,  it  is  advisable  from 
the  start  to  make  use  of  plate  cultures,  the  best  medium 
being  nutrient  agar  freshly  smeared  with  rabbit's  blood. 
The  sputum,  blood,  or  other  substance  to  be  examined 
is  streaked  across  several  plates  of  blood-smeared  agar, 
so  as  to  leave  on  some  considerable  of  the  material  and 
on  others  merely  the  slightest  trace.  An  easy  way  to 
get  blood  when  a  large  number  of  plates  are  to  be  made 
is  to  kill  a  rabbit  and  autopsy  it  immediately.  The 
skin  is  turned  back  from  the  chest  and  the  thorax 
opened  aseptically.  The  heart  is  cut  off  at  its  base 
and  dragged  over  some  twenty  to  forty  plates,  as  de- 
sired. The  blood  collecting  in  the  thorax  is  used  to 
smear  the  agar  in  a  number  of  tubes,  which  can  be  kept 
in  the  ice-chest  until  needed.  With  a  little  skill  blood 
.can  be  withdrawn  aseptically  from  the  ear  vein  of  a 
rabbit  by  means  of  a  glass  tube  armed  with  a  hypo- 
dermic needle. 

When  cultures  are  made  from  sputa  the  endeavor 
should  be  made  to  collect  the  expectoration  which  comes 
up  naturally,  so  as  not  to  get  any  more  than  necessary 
of  the  mouth  bacteria.  If  the  mouth  is  at  all  foul,  it 
should  be  cleansed  before  gathering  the  sputum.  Cul- 
tures should  be  made  as  soon  as  possible  after  obtaining 
the  material.  The  plates  are  put  in  the  incubator  for 


INFLUENZA  BACILLUS.  323 

eighteen  hours  and  then  examined  under  a  magnifica- 
tion of  about  100  diameters.  The  influenza  colonies, 
when  present,  will  be  found  in  the  neighborhood  of 
the  blood-cells,  much  lighter  in  hue,  somewhat  smaller, 
and  more  finely  granular  than  those  of  the  pneumococci. 
They  appear  scarcely  more  noticeable  than  the  groups  of 
blood-cells  that  have  lost  their  color  and  largely  disin- 
tegrated. With  higher  magnification  the  colonies  do  not 
show  the  individual  bacteria  distinctly,  and  thus  con- 
trast with  the  pneumococci.  The  suspicious  colonies  are 
fished  out,  inoculated  upon  blood  and  simple  nutrient 
agar,  and  examined  microscopically.  When  the  detec- 
tion of  the  bacilli  is  important,  and  there  are  any  puru- 
lent masses  in  the  sputum,  as  in  influenza  complicating 
phthisis,  these  are  washed,  as  under  directions  for  ex- 
amination of  sputa  for  mixed  infection  (page  306). 

On  1.5  per  cent,  sugar-agar  growth  also  occurs,  the 
colonies  appearing  as  extremely  small  droplets,  clear 
as  water,  often  only  recognizable  with  a  lens  (Pfeiffer). 
In  bouillon  a  very  scanty  development  takes  place,  un- 
less blood  is  added.  At  the  end  of  twenty-four  hours 
small,  white  particles  are  seen  on  the  surface,  which 
subsequently  sink  to  the  bottom,  forming  a  white, 
woolly  deposit,  while  the  bouillon  remains  clear. 

EESISTANCE  AND  LENGTH  OF  LIFE.  The  influenza 
bacillus  is  very  sensitive  to  desiccation;  a  pure  culture 
diluted  with  water  and  dried  is  destroyed  with  certainty 
in  twenty-four  hours;  in  dried  sputum  the  vitality, 
according  to  the  completeness  of  drying,  is  retained 
from  twelve  to  forty-eight  hours.  It  does  not  grow, 
but  soon  dies  in  water.  The  thermal  death-point  is 
60°  C.  with  five  minutes'  exposure  (Pfeiffer  and  Beck). 
In  bouillon  cultures  and  in  sputum  at  20°  C.  they  retain 


324  BACTERIOLOGY. 

their  vitality  for  from  a  few  days  to   two  or  three 
weeks. 

Pathogenesis.  The  bacillus  of  influenza,  in  so  far  as 
experiments  show,  produces  the  disease  only  in  monkeys 
and  to  a  less  extent  in  rabbits.  From  numerous  ex- 
periments made  by  Pfeiffer  on  guinea-pigs,  rats,  mice, 
and  pigeons  these  animals  seem  to  be  more  or  less  in- 
susceptible to  influenza.  When  a  small  quantity  of 
culture  on  blood-agar,  twenty-four  hours  old,  sus- 
pended in  1  c.c.  of  bouillon,  was  injected  intrave- 
nously into  rabbits,  Pfeiffer  found  that  a  characteristic 
pathogenic  effect  was  produced.  The  first  symptoms 
were  developed  in  one  and  a  half  to  two  hours  after 
the  injection.  The  animals  became  extremely  feeble, 
lying  flat  upon  the  floor,  with  their  limbs  extended, 
and  suffered  from  extreme  dyspnoea.  The  temperature 
rose  to  41°  C.  or  above.  At  the  end  of  five  or  six 
hours  they  were  able  to  sit  up  on  their  haunches  again, 
and  in  twenty-four  hours  had  recovered.  Larger  doses 
caused  the  death  of  the  animals  inoculated.  These 
results  are  attributed  by  Pfeiffer  to  toxic  products 
present  in  the  cultures,  and  in  none  of  his  experiments 
was  he  ever  able  to  obtain  effects  resembling  septicsemic 
infection.  In  some  of  the  experiments  on  monkeys, 
these  animals,  when  cultures  were  rubbed  into  the  nasal 
mucous  membrane,  showed  a  febrile  condition,  lasting 
for  a  few  days,  and  in  one  case  an  abscess  was  produced 
from  an  injection  into  the  subcutaneous  intercellular 
tissues:  but  in  no  instance  has  Pfeiffer  observed  a  mul- 
tiplication of  the  bacilli  introduced.  Recently  Cantani 
has  shown  that  it  is  possible  to  produce  an  infection  of 
influenza  in  rabbits  when  inoculated  with  small  doses 
(£  to  J  c.c.)  of  living  bacilli,  provided  the  point  of  least 


INFLUENZA  BACILLUS.  325 

resistance  is  chosen  for  the  inoculation — viz.,  the  brain, 
upon  which  the  toxic  products  of  the  bacillus  influenzae 
acts  most  powerfully. 

The  cell  bodies  of  the  bacilli  seem  to  possess  consid- 
erable pyogenic  action. 

Immunity.  Possibly  an  immunity  for  a  short  period 
against  the  influenza  poison  may  be  established  after  an 
attack.  At  least  in  three  experiments  made  by  Pfeiffer 
on  monkeys,  these  animals,  after  recovering  from  an 
inoculation  with  bacilli,  seemed  to  be  much  less  suscep- 
tible to  a  second  injection. 

In  patients  suffering  from  influenza  the  bacilli  are 
found  chiefly  in  the  nasal  and  bronchial  secretions. 
In  acute  uncomplicated  cases  they  may  be  observed 
microscopically  in  large  masses  and  often  in  absolutely 
pure  culture;  the  green,  purulent  sputum  derived  from 
the  bronchial  tubes  is  especially  suitable  for  examina- 
tion. The  older  the  process  is  the  fewer  bacilli  will  be 
found,  and  the  more  frequently  will  they  be  seen  lying 
within  the  pus -cells  instead  of  being  embedded  free  in 
the  secretion  as  at  first.  At  the  same  time  they  stain 
less  readily  and  present  more  irregular  and  swollen 
forms.  Very  frequently,  perhaps  almost  invariably 
(Finkler),  the  influenza  process  invades  portions  of  the 
lung  tissue.  In  severe  cases  a  form  of  pneumonia  is 
the  result,  which  is  lobular  and  purulent  in  character, 
and  accompanied  by  symptoms  almost  identical  with 
bronchopneumonia  due  to  the  pneumococcus.  The  walls 
of  the  bronchioles  and  alveolar  septa  become  densely 
infiltrated  with  leucocytes,  and  the  lumina  of  the  bron- 
chial tubes  and  alveoli  are  similarly  filled.  The  pus- 
cells  are  found  to  contain  more  or  less  influenza  bacilli. 
There  may  be  partial  softening  of  the  tissues,  or  even 


326  BACTERIOLOGY. 

abscess  formation.  Bacilli  are  found  in  fatal  cases 
to  have  penetrated  from  the  bronchial  tubes  not  only 
into  the  peribronchitic  tissue,  but  even  to  the  sur- 
face of  the  pleura,  and  rarely  they  have  been  obtained 
in  pure  cultures  in  the  purulent  exudation.  The  pleu- 
risy which  follows  influenza,  however,  is  usually  a 
secondary  infection,  due  to  the  streptococcus  or  pneu- 
mococcus.  Ordinarily  influenza  runs  an  acute  or  sub- 
acute  course,  and  not  infrequently  it  is  accompanied  by 
mixed  infections,  with  the  pneumococcns  and  the  strep- 
tococcus. Pfeiffer  was  the  first  to  draw  attention  to 
certain  chronic  conditions  depending  upon  the  influenza 
bacillus.  According  to  this  observer,  these  bacilli  may 
be  retained  in  the  lung  tissue  for  months  at  a  time, 
remaining  latent  awhile,  and  then  becoming  active 
again,  with  a  resulting  exacerbation  of  the  disease. 
Consumptives  are  particularly  susceptible  to  attacks  of 
influenza.  Williams,  in  the  examination  of  washed 
sputa  in  cases  of  pulmonary  tuberculosis,  has  on  numer- 
ous occasions  found  abundant  influenza  bacilli,  and  this 
in  the  summer,  when  no  influenza  was  known  to  be 
present  in  New  York.  Taken  together  with  Pfeiffer's 
results  in  Berlin,  this  indicates  that  at  all  times  of  the 
year  many  consumptives  carry  about  with  them  influ- 
enza bacilli,  and  that  very  likely  many  healthy  persons 
also  harbor  a  few.  Given  proper  climatic  conditions, 
we  have  at  all  times  the  seed  to  start  an  epidemic. 

The  influenza  bacillus  does  not  occur,  as  a  rule,  in 
the  blood.  According  to  Pfuhl  and  Nauwerck,  the 
influenza  bacilli  have  been  found  in  the  interior  organs 
and  the  brain,  but  these  observations  require  further 
confirmation.  So  far  as  positive  results  have  shown, 
influenza  would  seem  to  be  a  local  infection  confined  to 


INFLUENZA  BACILLUS.  327 

the  air-passages;  the  general  symptoms  produced  are 
due  probably  to  the  absorption  of  the  toxic  products  of 
the  specific  organism,  these  poisons  being  particularly 
active  in  their  effects  on  the  central  nervous  system. 

The  discovery  of  this  bacillus  enables  us  to  explain 
many  things,  previously  unaccountable,  in  the  cause  of 
epidemic  influenza.  We  now  know,  from  the  prop- 
erty of  the  influenza  bacillus  not  being  able  to  exist  for 
long  periods  in  dust,  that  the  disease  is  not  transmis- 
sible to  great  distances  through  the  air.  We  also 
know  that  the  infective  material  is  contained  only  in 
the  catarrhal  secretions.  Sporadic  cases,  or  the  sudden 
eruption  of  epidemics  in  any  localities  from  which  the 
disease  has  been  absent  for  a  long  time,  or  where  there 
has  been  no  new  importation  of 'infection,  may  possibly 
be  explained  by  the  fact  that  the  bacilli,  as  already 
mentioned,  often  remain  latent  in  the  lungs  or  bronchial 
secretions  of  the  body  for  many  months,  and  perhaps 
years,  and  then  become  active  again,  when  under  favor- 
able circumstances  they  may  be  communicated  to  others. 
The  bacteriological  diagnosis  of  influenza  is  of  consider- 
able importance  for  the  identification  of  clinically  doubt- 
ful cases,  which,  from  their  clinical  symptoms,  may  be 
mistaken  for  grippe,  or  vice  versa,  such  as  bronchitis, 
pneumonia,  or  tuberculosis.  Up  to  the  present  time, 
however,  the  diagnosis  gives  us  little  help  in  prognosis 
or  treatment. 

In  acute  uncomplicated  cases  the  probable  diagnosis 
can  be  frequently  made  by  microscopical  examinations 
of  stained  preparations  of  the  sputum,  there  being  pres- 
ent enormous  numbers  of  small  bacilli.  In  chronic  cases 
or  those  of  mixed  infection  the  culture  method  usually 
gives  a  positive  result.  The  bacillus  of  influenza  is  so 


328  BACTERIOLOGY. 

well  characterized  by  its  morphological,  staining,  and 
cultural  peculiarities  that  it  may  be  distinguished  with 
sufficient  certainty  for  practical  purposes  from  all  other 
bacteria  by  an  expert  bacteriologist  who  is  familiar 
with  it.  The  only  bacillus  which  resembles  it  at  all 
closely  is  the  pseudo-influenza  bacillus  found  by  Pfeiffer 
in  three  cases  of  bronchopneumonia.  This  bacillus  is 
culturally  very  similar  to  the  true  bacillus  influenzse, 
but  may  be  distinguished  from  it  by  its  larger  size  and 
tendency  to  grow  out  into  long  threads.  It  is  not  cer- 
tain but  that  it  is  a  form  of  the  influenza  bacillus. 
There  is  no  doubt  that  other  infections  are  also  included 
under  the  clinical  forms  of  influenza,  and  during  an 
epidemic  bronchopneumonias,  irregular  types  of  lobar 
pneumonias,  and  cases  of  bronchitis  frequently  have 
symptoms  so  closely  alike  that  the  nature  of  the  bac- 
teria active  in  the  case  is  very  frequently  different  from 
that  supposed  by  the  clinician.  Thus  in  four  consecu- 
tive autopsies  examined  by  the  writer  the  influenza 
bacillus  was  found  almost  in  pure  culture  in  one  case 
believed  to  be  due  to  the  pneumococcus,  and  entirely 
absent  in  two  of  the  three  believed  to  be  due  to  it. 
Except  for  these  examinations  the  clinician  would  be 
of  the  opinion  that  he  had  clearly  diagnosticated  bacte- 
riologically  the  cases,  while  in  fact  he  had  been  wrong 
in  three  of  the  four. 

The  striking  symptoms  in  acute  respiratory  diseases 
are  frequently  more  due  to  the  location  and  amount  of 
the  poisons  than  to  the  special  variety  of  organisms  pro- 
ducing them. 


CHAPTER  XXI. 

DIPHTHERIA   BACILLUS. 

History.  The  specific  contagious  disease  which  we 
now  call  diphtheria,  and,  therefore,  according  to  our 
present  belief,  the  bacilli  which  cause  it,  can  be  traced 
back  to  almost  the  Homeric  period  of  Grecian  history. 
The  Greeks  believed  that  it  had  been  communicated  to 
their  country  from  Egypt.  The  description  of  the 
pharyngeal  and  laryngeal  manifestations  of  this  dis- 
ease left  by  Aretseus  leaves  no  doubt  that  it  was  of 
diphtheria  that  he  wrote.  Galen,  in  his  remarks  on 
the  Chironion  ulcer,  tells  us  that  the  pseudomembrane 
was  gotten  rid  of  by  coughing  in  the  laryngeal  form 
of  the  disease,  and  by  hawking  in  the  pharyngeal  type. 
From  time  to  time  during  the  next  one  thousand  years 
we  hear  of  epidemics  both  in  Italy  and  in  other  por- 
tions of  the  civilized  world  which  indicate  that  the 
specific  bacteria  continued  to  be  handed  down  from 
case  to  case.  In  1517  we  read  of  a  malignant  form 
of  the  disease  raging  in  Switzerland,  along  the  Rhine, 
and  in  the  Netherlands.  In  1557  we  read  of  further 
epidemics  in  France,  Germany,  Holland,  and  Spain. 
The  disease  now  crossed  to  America,  and  in  the  New 
England  States  we  get  clear  accounts  of  its  ravages. 
Thus,  Samuel  Danforth,  in  1659,  Jost  four  of  his  eleven 
children  within  a  fortnight  by  a  ( '  malady  of  the  blad- 
ders in  the  windpipe/'  In  1765,  Home,  a  Scotchman, 


330  BACTERIOLOGY. 

tried  to  show  that  "  croup  "  and  pharyngeal  diphthe- 
ria were  different  diseases,  or,  in  bacteriological  terms, 
due  to  different  micro-organisms,  and  this  subject  re- 
mained under  controversy  until  it  was  recently  settled 
that  while  most  cases  were  undoubtedly  due,  at  least 
to  a  great  extent,  to  diphtheria  bacilli,  a  few  were  not. 

Bard,  an  American,  supported,  in  1771,  the  opposite 
theory  from  Home,  considering  the  process  the  same 
wherever  located.  In  this  ground  he  was  much  nearer 
to  the  facts  than  Home.  His  observations  upon  diph- 
theria were  very  important  and  accurate. 

In  1821,  Bretoaneau  published  his  first  essay  on  diph- 
theria in  Paris  and  gave  to  the  disease  its  present  name. 
His  observations  were  so  extensive  and  so  correct  that 
little  advance  in  knowledge  took  place  until  the  causal 
relations  of  the  diphtheria  bacilli  and  their  associated 
micro-organisms  to  the  disease  began  to  be  recognized. 
Since  then  the  combined  clinical,  bacteriological,  and 
pathological  studies  have  sufficed  to  make  diphtheria 
one  of  the  best  understood  of  diseases. 

The  Bacillus.  In  the  year  1883  bacilli  which  were 
very  peculiar  and  striking  in  appearance  were  shown 
by  Klebs  to  be  of  constant  occurrence  in  the  pseudo- 
membranes  from  the  throats  of  those  dying  of  true 
epidemic  diphtheria.  One  year  later  Loftier  published 
the  results  of  a  very  thorough  and  extensive  series  of 
investigations  on  this  subject.  He  found  the  bacillus 
described  by  Klebs  in  many  cases  of  throat  inflam- 
mations which  had  been  diagnosticated  as  diphthe- 
ria. He  separated  these  bacilli  from  the  other  bac- 
teria present  and  obtained  them  in  pure  culture.  When 
he  inoculated  the  bacilli  upon  the  abraded  mucous  mem- 
brane of  susceptible  animals,  more  or  less  characteristic 


DIPHTHERIA  BACILLUS.  331 

pseudomembranes  were  produced,  and  frequently  death 
or  paralysis  followed,  with  characteristic  lesions. 

In  1887  and  1888  further  studies  by  Loffler,  Eoux, 
and  Yersin  added  to  the  proof  of  the  dependence  of 
diphtheria  on  this  bacillus.  It  was  found  that  while 
no  other  forms  of  bacteria  were  constantly  met  with, 
the  diphtheria  bacilli  were  present  in  all  characteristic 
cases  of  diphtheria,  and  that  these  bacilli  possessed  the 
morphological,  cultural,  and  pathogenic  qualities  of 
those  described  by  Klebs  and  Loffler.  The  results  of 
these  investigations  have  since  been  confirmed  by  a 
great  number  of  combined  clinical  and  bacteriological 
observations  both  in  animals  and  human  beings.  A 
very  instructive  accidental  experiment  was  carried  out 
under  my  observation  some  years  ago.  One  of  the 
laboratory  workers  unintentionally  sucked  through  a 
defective  pipette  a  few  drops  into  the  mouth  of  a 
bouillon  culture  of  a  virulent  diphtheria  bacillus,  and 
two  days  later  characteristic  diphtheria  of  a  serious  type 
developed.  All  the  conditions  have  been  fulfilled  for 
diphtheria  which  are  necessary  to  the  most  rigid  proof 
of  the  dependence  of  an  infective  disease  upon  a  given 
micro-organism — viz.,  the  constant  presence  of  this 
organism  in  the  lesions  of  the  disease,  the  isolation  of 
the  organism  in  pure  culture,  the  reproduction  of  the 
essential  lesions  of  the  disease  in  animals  and  in  man 
by  inoculation  with  pure  cultures,  the  failure  to  produce 
all  the  characteristic  lesions  of  this  disease  by  any  other 
bacteria,  and  the  additional  proof  of  the  immunizing 
value  of  the  specific  substances  developed  in  animals 
subjected  to  injections  of  diphtheria  toxin.  In  view  of 
these  facts  we  are  now  justified  in  saying  that  the  name 
diphtheria,  or  at  least  primary  diphtheria,  should  be 


332 


BACTERIOLOGY. 


applied,  and  exclusively  applied,  to  that  acute  infectious 
disease  usually  associated  with  pseudomembranous  affec- 
tion of  the  mucous  membranes  which  is  primarily 
caused  by  the  bacillus  diphtherise  of  Loffler.  Other 
bacteria  do,  indeed,  occasionally  produce  lesions  which 
simulate  in  one  way  or  another  those  caused  by  the 
diphtheria  bacillus,  but  none  of  them  ever  produce 
lesions  similar  in  their  totality  to  those  of  a  charac- 
teristic case  of  diphtheria. 

Morphology.  When  cover-glass  preparations  made 
from  the  cultures  grown  on  blood-serum  are  examined 
the  diphtheria  bacilli  are  found  to  possess  the  following 
morphological  characteristics :  The  diameter  of  the 
bacilli  varies  from  0.2^  to  0.8/j.  and  the  length  usually 


FIG.  41. 


FIG.  42. 


One  of  very  characteristic  forms  ot 
diphtheria  bacilli  from  blood-serum 
cultures,  showing  clubbed  ends  and 
irregular  stain.  X  1100  diameters 
Stain,  methylene-blue. 


Extremely  long  form  of  diphtheria 
bacillus.  This  culture  has  grown  on 
artificial  media  for  four  years  and 
produces  strong  toxin.  X  1100  diam- 
eters. 


from  I/jt  to  6//,  but  exceptionally  even  longer  (see 
Fig.  42).  They  occur  singly  and  in  pairs  (see  Figs.  41 
to  44),  and  very  infrequently  in  chains  of  three  or  four. 
At  times,  especially  in  the  tissues,  branching  forms  are 


DIPHTHERIA  BACILLUS. 


333 


observed.  The  rods  are  straight  or  slightly  curved,  and 
usually  are  not  uniformly  cylindrical  throughout  their 
entire  length,  but  are  swollen  at  the  end  or  pointed  at 


FIG.  43. 


FIG.  44. 


Diphtheria  bacilli  characteristic  in 
shapes  but  showing  even  staining.  In 
appearance  similar  to  the  xerosis  ba- 
cillus. X  1100  diameters.  Stain,  me- 
thylene-blue. 


Non-virulent  diphtheria  bacilli, 
showing  stain  with  Neisser's  solu- 
tions, supposed  to  be  characteristic 
of  virulent  bacilli.  Bodies  of  bacilli 
in  smear,  faint  brown  ;  points,  dark 
blue. 


FIG.  45. 


Small  type  of  pseudodiphtheria  bacilli.    X  1000  diameters. 

the  ends  and  swollen  in  the  middle  portion.  The 
average  length  of  the  bacilli  in  cultures  from  different 
sources  frequently  varies  greatly,  and  even  from  the 


334  BACTERIOLOGY. 

same  culture  individual  bacilli  differ  much  in  their 
size  and  shape.  The  two  bacilli  of  a  pair  may  lie 
with  their  long  diameter  in  the  same  axis  or  at  an 
obtuse  or  an  acute  angle,  or  the  pairs  of  bacilli  may 
lie  side  by  side  or  irregularly  across  each  other.  The 
bacilli  possess  no  spores,  but  have  in  them  highly 
refractile  bodies  at  certain  stages  in  their  life. 

The  Klebs-Loffler  bacilli  stain  readily  with  ordinary 
aniline  dyes,  and  retain  fairly  well  their  color  after 
staining  by  Gram's  method.  When  Loffler's  alkaline 
solution  of  methylene-blue  is  applied  cold  for  five  min- 
utes or  warm  for  one  minute  the  bacilli  from  blood- 
serum  cultures  especially,  and  from  other  media  less 
constantly,  stain  in  an  irregular  and  extremely  charac- 
teristic way  (see  Fig.  41).  Many  of  the  bacilli  do  not 
stain  uniformly.  In  many  cultures  round  or  oval 
bodies  situated  at  the  ends  or  in  the  central  portions 
stain  much  more  intensely  than  the  rest  of  the  bacillus. 
Sometimes  these  highly  stained  bodies  are  thicker  than 
the  rest  of  the  bacillus;  again,  they  are  thinner  and 
surrounded  by  a  more  slightly  stained  portion.  The 
bacilli  seem  to  stain  in  this  peculiar  manner  at  a  cer- 
tain period  of  their  growth,  and  more  when  grown  on 
some  media  than  on  others,  so  that  only  a  portion  of 
the  organisms  taken  from  a  culture  at  any  one  time 
will  show  the  characteristic  staining.  In  old  cultures 
the  bacilli  stain  poorly  and  not  at  all  in  a  characteristic 
way.  The  same  round  or  oval  bodies  which  take  the 
methylene-blue  more  intensely  than  the  remainder  of 
the  bacillus  are  brought  out  still  more  distinctly  by  the 
Neisser  stain. 

The  Neisser  stain  is  carried  out  by  placing  the  cover- 
slip  smear  of  diphtheria  or  other  bacilli  in  solution  No. 


DIPHTHERIA  BACILLUS.  335 

1  for  from  two  to  three  seconds,  and  then,  after  washing, 
in  No.  2  for  from  three  to  five  seconds.  The  bacilli 
will  then  appear  either  entirely  brown  or  will  show  at 
one  or  both  ends  a  dark-blue  round  body.  With  char- 
acteristic diphtheria  bacilli  taken  from  a  twelve  to 
eighteen  hours'  growth  on  serum  nearly  all  will  show 
the  blue  bodies  (Fig.  44),  while  with  the  pseudotype 
(Fig.  45),  to  be  described  hereafter,  few,  if  any,  will 
be  seen. 

The  solutions  are  as  follows  : 

No  1. 

Alcohol  (96  per  cent. )      .         .         .  .  20  parts 

Methylene  blue  (Griibler)         .         .  .-       1  part 

Distilled  water          .        *.         .         .  .  950  parts. 

Acetic  acid  (glacial)          ...  .  50     "• 

No.  2. 

Bismark  brown         .         .         .         .         .         1  part. 
Boiling  distilled  water      ...         .     500  parts. 

The  Neisser  stain  has  been  advocated  in  order  to 
separate  the  virulent  from  the  non-virulent  bacilli 
without  the  delay  of  inoculating  animals;  but  in  our 
hands,  with  a  very  large  experience,  neither  the  Neisser 
stain  nor  other  stains,  such  as  the  modifications  of  the 
Roux  stain,  have  given  any  more  information  as  to  the 
virulence  of  the  bacilli  than  the  usual  methylene-blue 
solution  of  Loffler.  A  small  percentage  of  virulent 
bacilli  fail  to  take  the  Neisser  stain,  and  quite  a  few 
non-virulent  psetidodiphtheria  bacilli  show  the  dark 
bodies.  In  New  York  there  are  also  a  large  number 
of  bacilli  which  seem  to  have  all  the  staining  and  cul- 
tural characteristics  of  the  virulent  bacilli,  and  yet  are 
non-virulent  in  the  sense  that  they  produce  no  specific 


336  BACTERIOLOGY. 

toxin.  To  one  who  is  accustomed  to  the  Loffler  stain 
it  gives  as  much  information  as  any  other  as  to  the 
specific  virulence  of  the  bacilli.  The  Neisser  stain  will 
undoubtedly  cause  the  examiner  to  suspect  more  strongly 
some  bacilli  of  being  virulent  than  the  Loffler  stain, 
but  with  the  varieties  met  with  in  New  York  this  sus- 
picion is  as  apt  to  be  wrong  as  right.  As  will  be  stated 
more  fully  later,  nothing  but  the  animal  inoculations 
with  control  injections  of  antitoxin  will  separate  spe- 
cifically virulent  from  non-virulent  bacilli. 

The  morphology  of  the  diphtheria  bacillus  varies  con- 
siderably with  the  different  culture  media  employed. 
On  glycerin  agar  or  simple  nutrient  agar  it  is  smaller, 
and,  as  a  rule,  more  regular  in  form  than  when  grown 
on  other  usual  culture  media  (Fig.  46).  Short,  spindle, 

PIG.  46. 


Diphtheria  bacilli  from  agar  culture.    X  1000  diameters. 

lancet,  or  club-shaped  forms,  staining  uniformly,  are 
here  commonly  observed.  The  bacilli  which  have  de- 
veloped in  the  pseudomembranes  or  exudate  in  cases  of 
diphtheria  resemble  in  shape  those  grown  on  blood- 
serum,  but  stain  more  evenly. 


DIPHTHERIA  BACILLUS.  337 

But  though  the  morphology  of  the  diphtheria  bacillus 
is  more  regular  under  some  circumstances  than  others, 
its  chief  morphological  characteristic  is  its  irregularity 
of  form  and  size. 

Biology.  The  Klebs-Loffler  bacillus  is  non-motile 
and  non-liquefying.  It  is  aerobic.  It  grows  most 
readily  in  the  presence  of  oxygen,  but  also  without 
it;  it  is  thus  facultative  anaerobic.  It  does  not  form 
spores.  Its  thermal  death-point  with  ten  minutes' 
exposure  is  about  58°  C.,  and  with  longer  exposure  a 
lower  temperature;  it  is  more  easily  destroyed  by  dis- 
infectants than  many  other  bacteria.  In  the  dry  state 
and  exposed  to  diffuse  light  diphtheria  bacilli  usually 
die  in  a  few  days  or  may  live  for  weeks  or  months; 
when  in  the  dark,  or  protected  by  a  film  of  mucus  or 
albumin,  they  may  live  for  even  longer  periods.  Thus 
I  found  scrapings  from  a  dry  bit  of  membrane  to 
contain  vigorous  and  virulent  living  bacilli  for  a 
period  of  four  months  after  removal  from  the  throat, 
and  if  the  membrane  had  not  been  at  that  time  com- 
pletely used,  living  bacilli  could  probably  have  been 
obtained  for  a  much  longer  period;  in  culture  media 
when  kept  at  the  blood  heat  they  usually  die  after  a 
few  weeks,  but  under  certain  conditions,  as  when  sealed 
in  tubes  and  protected  from  heat  and  light,  they  retain 
their  virulence  for  years.  The  bacillus  is  not  sensitive 
to  cold,  for  I  found  it  to  retain  its  virulence  after  ex- 
posure for  two  hours  to  several  hundred  degrees  below 
zero.  It  begins  to  develop,  but  grows  slowly,  at  a 
temperature  of  20°  C.,  or  even  less.  It  grows  more 
rapidly  as  the  temperature  rises,  and  attains  its  maximum 
development  at  37°  C.  It  may  grow  at  a  temperature 
as  high  as  41°  C.  and  retain  its  virulence  for  months. 

22 


338  BACTERIOLOGY. 

Growth  on  Blood-serum.  Blood-serum,  especially  in 
the  form  of  L6 frier's  mixture,  is  the  most  favorable 
medium  for  the  growth  of  the  diphtheria  bacillus, 
and  is  used  particularly  for  diagnostic  purposes  in 
examining  cultures  from  the  throats  of  persons  sus- 
pected of  having  diphtheria.  For  its  preparation, 
•see  p.  377.  If  we  examine  the  growth  of  the  diph- 
theria bacillus  in  pure  culture  on  blood-serum  we  shall 
find  at  the  end  of  from  eight  to  twelve  hours  small  colo 
nies  of  bacilli,  which  appear  as  pearl-gray,  whitish-gray, 
or,  more  rarely,  yellowish-gray,  slightly  raised  points. 
The  colonies  when  separated  from  each  other  may  in- 
crease in  forty-eight  hours  so  that  the  diameter  may 
be  one-eighth  of  an  inch.  The  borders  are  usually 
somewhat  uneven.  The  colonies  lying  together  be- 
come confluent  and  fuse  into  one  mass,  when  the 
serum  is  moist.  During  the  first  twelve  hours  the 
colonies  of  the  diphtheria  bacilli  are  about  equal  in  size 
to  those  of  the  other  pathogenic  bacteria  which  are  often 
present  in  the  throat;  but  after  this  time  the  diphtheria 
colonies  become  larger  than  those  of  the  streptococci  and 
smaller  than  those  of  the  staphylococci.  The  diph- 
theria bacilli  in  their  growth  never  liquefy  the  blood- 
serum. 

Growth  on  Agar.  On  1  per  cent,  slightly  alkaline, 
plain  nutrient  or  glycerin-agar  the  growth  of  the 
diphtheria  bacillus  is  less  certain  and  luxuriant  than 
upon  blood-serum;  but  the  appearance  of  the  colonies 
when  examined  under  a  low-power  lens,  though  very 
variable,  is  often  far  more  characteristic.  (See  Fig.  30, 
page  229,  and  Fig.  47,  page  339.)  The  diphtheria 
bacillus  obtained  from  cultures  which  have  developed 
for  some  time  on  culture  media  grows  well,  as  a  rule, 


DIPHTHERIA  BACILLUS.  339 

on  suitable  nutrient  or  glycerin-agar,  but  when  fresh 
from  pseudomembranes  it  frequently  grows  on  these 
media  with  great  difficulty,  and  the  colonies  develop 
so  slowly  as  to  be  covered  up  by  the  more  luxuriant 
growth  of  other  bacteria,  or  fail  to  develop  at  all. 

FIG.  47. 


Colonies  of  diphtheria  bacilli.    X  200  diameters. 

If  the  colonies  develop  deep  in  the  substance  of  the 
agar  they  are  usually  round  or  oval,  and,  as  a  rule,  pre- 
sent no  extensions;  but  if  near  the  surface,  commonly 
from  one,  but  sometimes  from  both  sides,  they  spread 
out  an  apron-like  extension  which  exceeds  in  surface 
area  the  rest  of  the  colony.  When  the  colonies  develop 
entirely  on  the  surface  they  are  more  or  less  coarsely 
granular,  and  usually  have  a  dark  centre  and  vary 
very  much  in  their  thickness.  Some  are  almost  trans- 
lucent; others  are  thick  and  almost  as  luxuriant  as  the 
staphylococcus.  The  edges  are  sometimes  jagged  and 
frequently  shade  off  into  a  delicate  lace  like  fringe;  at 
other  times  the  margins  are  more  even  and  the  colonies 


340  BACTERIOLOGY. 

are  nearly  circular.  With  a  high-power  lens  the  edges 
show  sprouting  bacilli.  The  colonies  are  gray  or 
grayish-white  by  reflected  light  and  pure  gray  with 
an  olive  tint  by  transmitted  light. 

The  growth  of  the  diphtheria  bacillus  upon  agar 
presents  certain  peculiarities  which  are  of  practical 
importance.  If  a  large  number  of  the  bacilli  from  a 
recent  culture  are  implanted  upon  a  properly  prepared 
agar  plate  a  certain  and  fairly  vigorous  growth  will 
always  take  place.  If,  however,  the  agar  is  inoculated 
with  an  exudate  from  the  throat  which  contains  but 
few  bacilli,  no  growth  whatever  may  occur,  while  the 
tubes  of  coagulated  blood-serum  inoculated  with  the 
same  exudate  contain  the  bacilli  abundantly.  Again, 
agar  prepared  from  broth  made  from  different  speci- 
mens of  beef  or  to  which  different  peptones  have  been 
added,  varies  as  to  its  suitability  for  the  growth  of  the 
bacilli.  Because  of  the  uncertainty,  therefore,  of  ob- 
taining a  growth  by  the  inoculation  of  agar  with  bacilli 
unaccustomed  to  this  medium,  agar  is  a  far  less  reliable 
medium  than  blood-serum  for  use  in  primary  cultures 
for  diagnostic  purposes.  If  used  the  agar  should  at 
least  be  tested  by  means  of  a  culture  before  being  em- 
ployed. A  mixture  composed  of  two  parts  of  a  1J 
per  cent,  nutrient  agar  and  one  part  of  sterile  ascitic 
fluid  makes  a  medium  upon  which  the  bacillus  grows 
much  more  luxuriantly  but  not  so  characteristically. 
The  mixture  is  made  by  adding  the  warmed  ascitic 
fluid  to  the  tubes  containing  the  melted  agar  cooled  to 
60°.  After  shaking  the  Petri  plates  are  filled. 

The  Isolation  of  the  Diphtheria  Bacillus  from  Plate 
Cultures.  Nutrient  plain  or  glycerin-agar,  with  or 
without  the  addition  of  ascitic  fluid,  is,  however,  the 


DIPHTHERIA  BACILLUS.  341 

medium  employed  to  get  by  plating  methods  a  pure 
culture  from  the  original  serum  tube.  The  agar  should 
be  freshly  melted  and  poured  in  the  Petri  dish  for  this 
purpose.  After  it  has  hardened  the  layers  in  a  number 
of  plates  are  streaked  across  with  bacteria  from  colonies 
on  the  serum  culture,  which  appear  in  size  and  color 
like  the  diphtheria  bacilli.  Other  plates  are  made  from 
a  general  mixture  of  all  the  bacteria,  selected,  as  a  rule, 
from  the  drier  portion  of  the  serum.  The  plates  are  left 
in  the  incubator  for  twelve  hours  at  37°  C.  In  the 
examination  of  the  plates  one  should  first  seek  for 
typical  colonies  and  then  later  for  any  that  look  nearest 
the  characteristic  picture.  Diphtheria  colonies  are  very 
apt  to  be  found  at  the  edges  of  the  streaks  of  bacterial 
growth. 

Growth  in  Bouillon.  The  diphtheria  bacillus  usu- 
ally grows  readily  in  broth  slightly  alkaline  to  litmus. 
The  characteristic  growth  in  neutral  bouillon  is  one 
showing  fine  grains.  These  deposit  along  the  slles  and 
bottom  of  the  tube,  leaving  the  broth  nearly  clear.  A 
few  cultures  in  neutral  bouillon  and  many  in  alkaline 
bouillon  produce  for  twenty-four  or  forty-eight  hours 
a  more  or  less  diffuse  cloudiness,  and  frequently  a  film 
forms  over  the  surface  of  the  broth.  On  shaking  the 
tube  this  film  breaks  up  and  slowly  sinks  to  the  bottom. 
This  film  is  more  apt  to  develop  during  the  growth  of 
cultures  which  have  long  been  cultivated  in  bouillon,  and 
indeed  after  a  time  the  entire  development  may  appear 
on  the  surface  in  the  form  of  a  friable  pedicle.  The 
diphtheria  bacillus  in  its  growth  causes  a  fermentation 
of  the  meat  sugars  and  the  glucose,  and  thus  changes 
the  reaction  of  the  bouillon,  rendering  it  distinctly  less 
alkaline  within  forty-eight  hours,  and  then,  after  a  vari- 


342  BACTERIOLOGY. 

able  time,  when  all  the  fermentable  sugars  have  been 
decomposed,  more  alkaline  again  through  the  progress- 
ing fermentation  of  other  substances.  Among  the 
products  formed  by  its  growth  is  the  diphtheria  toxin. 

Growth  in  Ascitic  Bouillon.  Many  diphtheria  bacilli 
grow  but  feebly  in  nutrient  bouillon  when  first  re- 
moved from  the  throat.  These  develop  more  luxuri- 
antly when  to  the  bouillon  25  per  cent,  aseitic  fluid  or 
blood-serum  is  added. 

Growth  on  Gelatin.  The  growth  on  this  medium 
is  much  slower,  more  scanty,  and  less  characteristic  than 
that  on  the  other  media  mentioned,  on  account  of  the 
lower  temperature  at  which  it  is  used. 

Growth  in  Milk.  The  diphtheria  bacillus  grows 
readily  in  milk,  beginning  to  develop  at  a  compara- 
tively low  temperature  (20°  C.).  Thus  milk  having 
become  inoculated  with  the  bacillus  from  some  cases  of 
diphtheria  may  under  certain  conditions  be  the  means 
of  conveying  infection  to  previously  healthy  persons. 
Though  this  growth  takes  place,  the  milk  remains  un- 
changed in  appearance. 

Pathogenesis.  The  diphtheria  bacillus  is  pathogenic 
for  guinea-pigs,  rabbits,  chickens,  pigeons,  small  birds, 
and  cats;  also  in  a  lesser  degree  for  dogs,  goats,  cattle, 
and  horses,  but  hardly  at  all  for  rats  and  mice.  In 
spite  of  its  pathogenic  qualities  for  these  animals  true 
diphtheria  occurs  in  them  with  extreme  rarity.  As  a 
rule,  supposed  diphtheritic  inflammations  in  them  are 
due  to  other  bacteria  which  cannot  produce  the  disease 
in  man. 

The  virulence  of  diphtheria  bacilli  from  different 
sources,  as  measured  by  their  toxin  production,  varies 
enormously.  Thus  0.002  c.c.  of  a  forty-hour  bouillon 


DIPHTHERIA  BACILLUS.  343 

culture  of  one  bacillus  will  kill  a  guinea-pig,  while  it 
would  require  1  c.c.  of  the  culture  of  another  bacillus 
to  kill.  The  same  marked  variation  occurs  in  the 
amount  of  toxin  produced  by  different  bacilli  in  their 
growth  in  media  outside  of  the  body.  There  are  also 
bacilli  which  produce  no  specific  toxin  whatever  and 
yet  appear  to  have  all  the  other  characteristics  of  viru- 
lent bacilli.  Moreover,  the  diphtheria  bacilli  differ 
greatly  in  the  tenacity  with  which  they  retain  their 
virulence  when  grown  outside  the  body.  The  bacillus 
that  we  have  used  in  the  laboratory  of  the  health  de- 
partment has  retained  its  virulence  unaltered  for  four 
years  in  frequently  renewed  bouillon  cultures.  Other 
bacilli  have  lost  50  per  cent,  of  their  virulence  after 
being  kept  for  only  a  few  months.  The  passage  of 
diphtheria  bacilli  through  the  bodies  of  susceptible 
animals  does  not  increase  their  virulence  to  any  con- 
siderable extent,  this  being  probably  due  to  the  fact 
that  the  bacilli  multiply  but  little  in  the  tissues. 

At  the  autopsy  of  animals  dying  from  the  poisons 
produced  by  the  bacilli  the  characteristic  lesions  de- 
scribed by  Loftier  are  found.  At  the  seat  of  inocula- 
tion there  is  a  grayish  focus  surrounded  by  an  area  of 
congestion;  the  subcutaneous  tissues  for  some  distance 
around  are  oedematous;  the  adjacent  lymph-nodes  are 
swollen;  and  the  serous  cavities,  especially  the  pleural 
and  the  pericardial,  frequently  contain  an  excess  of 
fluid,  usually  clear,  but  at  times  turbid;  the  lungs  are 
generally  congested.  In  the  organs  are  found  numer- 
ous smaller  and  larger  masses  of  necrotic  cells,  which 
are  permeated  by  leucocytes.  The  heart  and  voluntary 
muscular  fibres  usually  show  degenerative  changes. 
Occasionally  there  is  fatty  degeneration  of  the  liver  and 


344  BACTERIOLOGY. 

kidneys.  The  number  of  leucocytes  in  the  blood  is  in- 
creased. From  the  area  surrounding  the  point  of  inocu- 
lation virulent  bacilli  may  be  obtained,  but  in  the  in- 
ternal organs  they  are  only  occasionally  found,  unless  an 
enormous  number  of  bacilli  have  been  injected.  Paral- 
ysis, commencing  usually  in  the  posterior  extremities 
and  then  gradually  extending  to  other  portions  of  the 
body  and  causing  death  by  paralysis  of  the  heart  or 
respiratory  organs,  is  also  produced  in  many  cases  in 
which  ihe  inoculated  animals  do  not  succumb  to  a  too 
rapid  intoxication.  In  rare  instances  the  muscles  of 
the  neck  or  of  the  larynx  are  first  paralyzed,  and  thus 
characteristic  symptoms  are  caused.  In  a  number  of 
animals  I  have  seen  recovery  take  place  three  to  six 
weeks  after  the  onset  of  the  paralysis. 

Diphtheria  Toxin.  It  is  evident  that  a  micro  organ- 
ism which,  when  injected  subcutaneonsly,  destroys  the 
life  of  susceptible  animals  and  produces  such  marked 
anatomical  changes  in  the  internal  organs,  while  it  is 
found  only  at  or  near  the  point  of  inoculation,  must 
owe  its  pathogenic  power  to  the  formation  of  a  poison 
which,  being  absorbed,  gives  rise  to  toxaemia  and  death. 
This  poison  or  toxin  has  been  partially  isolated  by  Roux 
and  Yersin,  and  others,  by  filtration  through  porous 
porcelain  from  cultures  of  the  living  bacilli.  It  has  not 
yet  been  successfully  analyzed,  so  that  its  chemical 
composition  is  unknown,  but  it  has  many  of  the  prop- 
erties of  proteid  substances,  and  can  well  be  designated 
by  the  term  active  proteid  (see  page  72).  Diphtheria 
toxin  is  totally  destroyed  by  boiling  for  five  minutes, 
and  loses  some  95  per  cent,  of  its  strength  when  ex- 
posed to  75°  C.  for  the  same  time  ;  73°  C.  destroys 
only  about  85  per  cent,  and  60°  very  little.  Lower 


DIPHTHERIA  BACILLUS.  345 

temperatures  only  alter  it  very  gradually.  Kept  from 
light  and  air  and  in  cold  storage  it  keeps  almost  un- 
altered for  years. 

The  Production  of  Toxin  in  Culture  Media.  The  arti- 
ficial production  of  toxin  in  cultures  of  the  diphtheria 
bacillus  has  been  found  to  depend  upon  definite  condi- 
tions, which  are  of  practical  importance  in  obtaining 
toxin  for  the  inoculation  of  horses,  and  also  of  theoret- 
ical interest  in  explaining  why  cases  of  apparently  equal 
local  severity  have  such  different  degrees  of  toxic  ab- 
sorption. The  researches  of  Roux  and  Yersin  laid  the 
foundation  of  our  knowledge.  Their  investigations  have 
been  continued  by  Theobald  Smith,  Spronck,  ourselves, 
and  others.  After  an  extensive  series  of  investigations 
we  (Park  and  Williams)  came  to  the  following  conclu- 
sions :  Toxin  is  produced  by  fully  virulent  diphtheria 
bacilli  at  all  times  during  their  life  when  the  conditions 
are  favorable.  Under  less  favorable  conditions  some 
bacilli  are  able  to  produce  toxin  while  others  are  not; 
or  it  may  be  that  some  conditions  favor  some  bacilli 
while  they  are  deleterious  to  others.  Diphtheria  ba- 
cilli may  find  conditions  suitable  for  luxuriant  growth, 
but  unsuitable  for  the  production  of  toxin.  The  re- 
quisite conditions  for  a  good  development  of  toxin,  as 
judged  by  the  behavior  of  a  number  of  cultures,  are  a 
temperature  from  about  35°  to  37.5°  C.,  a  suitable  cul- 
ture medium,  such  as  a  2  per  cent,  peptone  nutrient 
bouillon  of  an  alkalinity  which  should  be  about  8  c.c. 
of  normal  soda  solution  per  litre  above  the  neutral 
point  to  litmus,  and  prepared  from  a  suitable  peptone 
and  meat.  The  culture  fluid  should  be  in  compara- 
tively thin  layers  and  in  large-necked  Erlenmeyer 
flasks,  so  as  to  allow  of  a  free  access  of  air.  The 


346  BACTERIOLOGY. 

greatest  accumulation  of  toxin  in  bouillon  is  after  a 
duration  of  growth  of  the  culture  of  from  five  to  ten 
days,  according  to  the  peculiarities  of  the  culture  em- 
ployed. At  a  too  early  period  toxin  has  not  sufficiently 
accumulated,  at  a  too  late  period  it  has  begun  to  de- 
generate. In  our  experience  the  amount  of  muscle 
sugar  present  in  the  meat  makes  no  appreciable  differ- 
ence in  the  toxin  produced,  so  long  as  the  bouillon  has 
been  made  sufficiently  alkaline  to  prevent  the  acid 
produced  by  the  fermentation  of  the  sugar  from  pro- 
ducing in  the  bouillon  an  acidity  sufficient  to  inhibit  the 
growth  of  the  bacilli.  In  neutral  bouillon,  as  pointed 
out  by  Smith  and  Spronck,  the  sugar  does  produce  suffi- 
cient acid  to  interfere  with  the  growth  of  the  bacilli 
and  the  development  of  toxin.  This  can  be  prevented 
by  the  previous  destruction  of  the  sugar  through  the 
fermentation  caused  by  the  growth  of  the  colon  bacilli. 
After  the  fermentation  0.1  per  cent,  of  glucose  should 
be  added.  Beside  the  sugar  and  allied  bodies  in  the 
meat  there  are  other  substances,  whose  nature  is  un- 
known, which  hinder  or  aid  a  full  growth  of  the  bacilli 
or  production  of  toxin.  This  is  true  of  bouillon  made 
directly  from  fresh  meat,  fermented  meat,  or  meat  ex- 
tracts. With  the  meat  as  we  obtain  it  in  New  York  we 
get  better  results  with  uufermented  meat  than  with  fer- 
mented. In  Boston,  with  the  same  bacillus,  Smith 
gets  more  toxin  from  the  fermented  bouillon.  Con- 
tradictory results  have  been  obtained  by  others,  and 
must  be  attributed  to  the  difference  in  the  materials  used. 
Under  the  best  conditions  we  can  devise,  toxin  begins 
to  be  produced  by  bacilli  from  some  cultures  when 
freshly  sown  in  bouillon  some  time  during  the  first 
twenty-four  hours;  from  other  cultures,  for  reasons  not 


DIPHTHERIA  BACILLUS.  347 

well  understood,  not  for  from  two  to  four  days.  In 
neutral  bouillon  the  culture  fluid  frequently  becomes 
slightly  acid  and  toxin  production  may  be  delayed  for 
from  one  to  three  weeks.  The  greatest  accumulation 
of  toxin  is  on  the  fourth  day,  on  the  average,  after  the 
rapid  production  of  toxin  has  commenced.  After  that 
time  the  number  of  living  bacilli  rapidly  diminishes 
in  the  culture,  and  the  conditions  for  those  remaining 
alive  are  not  suitable  for  the  rapid  production  of  toxin. 
As  the  toxin  is  not  stable,  the  deterioration  taking  place 
in  the  toxin  already  produced  is  greater  than  the  amount 
of  new  toxin  still  forming. 

Bacilli,  when  repeatedly  transplanted  from  bouillon 
to  bouillon,  gradually  come  to  grow  on  the  surface 
only.  This  characteristic  seems  to  aid  in  the  develop- 
ment of  toxin. 

The  relations  of  toxin  to  antitoxin  will  be  described 
after  the  subject  of  antitoxin  has  been  considered. 

Non-virulent  Diphtheria  Bacilli.  Xerosis  Bacilli.  In 
the  very  large  number  of  tests  for  virulence  of  the 
bacilli  obtained  from  hundreds  of  cases  of  suspected 
diphtheria  which  have  been  carried  out  during  the 
past  six  years  in  the  laboratories  of  the  Health  De- 
partment of  New  York  City,  in  over  95  per  cent,  of 
cases  the  bacilli  derived  from  exudates  or  pseudomem- 
branes  and  possessing  the  characteristics  of  the  Loffler 
bacilli  have  been  found  to  be  virulent,  that  is  producers 
of  diphtheria  toxin.  But  there  are,  however,  in  inflamed 
throats  as  well  as  in  healthy  throats,  either  alone  or 
associated  with  the  virulent  bacilli,  occasionally  bacilli, 
which  though  morphologically  and  in  their  behavior  on 
culture  media  identical  with  the  Klebs-Loffler  bacillus, 
yet  producers,  at  least  in  artificial  culture  media  and 


348  BACTERIOLOGY. 

the  usual  test  animals,  of  no  appreciable  diphtheria 
toxin.  Between  bacilli  which  produce  a  great  deal 
of  toxin  and  those  which  apparently  produce  none 
we  find  all  grades  of  virulence.  We  believe,  there- 
fore, that  in  accordance  with  Roiix  and  Yersiri  these 
bacilli  should  be  considered  as  attenuated  varieties 
of  the  diphtheria  bacillus  which  have  lost  their  power 
to  produce  diphtheria  toxin.  These  observers,  and 
others  following  them,  have  shown  that  the  virulent 
bacilli  can  be  artificially  attenuated  by  cultivating  them 
at  a  temperature  of  39.5°  to  40°  C.  in  a  current  of  air. 
So  far  as  we  know,  bacilli  which  produce  no  specific 
toxin  have  never  later  been  found  to  develop  it.  In  our 
experience  some  cultures  hold  their  virulence  even  when 
grown  at  41°  C.  for  a  number  of  months,  while  others 
lose  it  more  quickly.  Bacilli  are  also  found  which 
resemble  diphtheria  bacilli  very  closely  except  in  toxin 
production,  but  differ  in  one  or  more  particulars.  Both 
these  and  the  characteristic  non  virulent  bacilli  are  found 
occasionally  upon  all  the  mucous  membranes,  both  when 
inflamed  and  when  apparently  normal.  From  varieties 
of  this  sort  having  been  found  in  a  number  of  .cases 
of  the  condition  known  as  xerosis  conjunctivas  by 
Kuschbert  and  Neisser,  these  bacilli  are  often  called 
xerosis  bacilli.  Under  this  name  different  observers 
have  placed  bacilli  identical  with  the  diphtheria  bacilli 
and  others  differing  quite  markedly  from  them.  Fig. 
43,  though  taken  from  virulent  bacilli,  gives  an  exact 
picture  of  many  of  the  xerosis  variety.  These  bacilli 
may  be  almost  non-pathogenic  in  guinea-pigs,  or  they 
may  kill,  as  we  have  found  in  a  number  of  instances, 
in  doses  of  2  to  5  c.c.  hypodermatically  injected.  Ani- 
mals are  not  protected  by  diphtheria  antitoxin  from 


DIPHTHERIA  BACILLUS.  349 

the  action  of  these  bacilli.  At  autopsy  the  bacilli  are 
usually  found  more  or  less  abundantly  in  the  blood  and 
internal  organs.  In  this  very  same  location,  however, 
diphtheria  bacilli  are  found  of  very  low  toxic  power, 
so  that  here,  again,  we  cannot  assert  that  these  xerosic 
bacilli  have  not  come  from  true  diphtheria  stock. 

Location  of  Diphtheritic  Inflammations  and  Viru- 
lence of  Bacilli.  Virulent  bacilli  produce  and  are 
found  not  only  in  pseudomembranous  inflammations 
of  the  fauces,  larynx,  and  nasal  cavities,  but  also  occa- 
sionally in  membranous  affections  of  the  skin,  vagina, 
rectum,  conjunctiva,  nose,  and  ear  (simple  mem- 
branous rhinitis  and  otitis  media).  From  the  severity 
of  an  isolated  case  the  virulence  of  the  bacilli  cannot  be 
accurately  determined.  The  most  virulent  bacillus  I 
have  ever  found  was  obtained  from  a  mild  case  of  diph- 
theria simulating  tonsillitis.  Another  case,  however, 
infected  by  this  bacillus  proved  to  be  very  severe.  In 
localized  epidemics  the  average  severity  of  the  cases 
probably  indicates  roughly  the  virulence  of  the  bacillus 
causing  the  infection,  as  here  the  individual  susceptibil- 
ity of  the  different  persons  infected  would,  in  all  likeli- 
hood, when  taken  together,  be  similar  to  that  of  other 
groups;  but  even  in  this  instance  special  conditions  of 
climate,  food,  or  race  may  influence  certain  localities. 
Moreover,  the  bacteria  associated  with  the  diphtheria 
bacilli,  and  which  are  liable  to  be  transmitted  with  them, 
may  influence  the  severity  of  and  the  complications 
arising  in  the  cases. 

Virulent  Bacilli  in  Healthy  Throats.  Fully  virulent 
bacilli  have  frequently  been  found  in  healthy  throats 
of  persons  who  have  been  brought  in  direct  contact 
with  diphtheria  patients  or  infected  clothing  without 


350  BACTERIOLOGY. 

contracting  the  disease.  It  is,  therefore,  apparent  that 
infection  in  diphtheria,  as  in  other  infectious  diseases, 
requires  not  only  the  presence  of  virulent  bacilli,  but 
also  a  susceptib  lity  to  the  disease,  which  may  be 
inherited  or  acquired.  Among  the  predisposing  in- 
fluences which  contribute  to  the  production  of  diph- 
theritic infection  may  be  mentioned  the  breathing  of 
foul  air  and  living  in  overcrowded  and  ill-ventilated 
rooms,  poor  food,  certain  diseases,  more  particularly 
catarrhal  inflammations  of  the  mucous  membranes,  and 
depressing  conditions  generally.  Under  these  condi- 
tions an  infected  mucous  membrane  may  become  sus- 
ceptible to  disease.  In  connection  with  Beebe  (1894) 

1  made  an  examination  of  the  throats  of  330  healthy 
persons  who  had  not  come  in  contact,  so  far  as  known, 
with  diphtheria,  and  we  found  virulent  bacilli  in  8  only, 

2  of  whom  later  developed  the  disease.    In  24  of  the  330 
healthy  throats  non-virulent  bacilli  or  attenuated  forms 
of  the  diphtheria  bacillus  were  found.      Very  similar 
observations  have  been  made  by  others  in  many  widely 
separated  countries. 

The  Persistence  of  Diphtheria  Bacilli  in  the  Throat. 
The  continued  presence  of  virulent  diphtheria  bacilli 
in  the  throats  of  patients  who  have  recovered  from  the 
disease,  and  after  the  disappearance  of  the  exndate,  has 
been  repeatedly  demonstrated.  Beebe  and  I  found  that 
in  304  of  605  consecutive  cases  the  bacilli  disappeared 
within  three  days  after  the  disappearance  of  the  pseudo- 
membrane;  in  176  cases  they  persisted  for  seven  days, 
in  64  cases  for  twelve  days,  in  36  cases  for  fifteen  days, 
in  12  cases  for  three  weeks,  in  4  cases  for  four  weeks, 
and  in  2  cases  for  nine  weeks.  Since  then  1  have  met 
with  a  case  in  which  they  persisted  for  six  months. 


DIPHTHERIA  BACILLUS.  351 

Pseudodiphtheria  Bacilli.  Beside  the  typical  baciili 
which  produce  diphtheria  toxin  and  those  which  do  not, 
but  which,  so  far  as  we  can  determine,  are  otherwise  iden- 
tical with  the  Loffler  bacillus,  there  are  other  bacilli 
found  in  positions  similar  to  those  in  which  diphtheria 
bacilli  abound,  which,  though  resembling  these  organ- 
isms in  many  particulars,  yet  differ  from  them  as  a  class 
in  others  equally  important.  The  variety  most  preva- 
lent is  rather  short,  plump,  and  more  uniform  in  size 
and  shape  than  the  true  Loffler  bacillus  (Fig.  45).  On 
blood-serum  their  colony  growth  is  very  similar  to  that 
of  the  diphtheria  bacilli.  The  great  majority  of  them 
in  any  culture  show  no  polar  granules  when  stained 
by  the  Neisser  method,  and  stain  evenly  throughout 
with  the  alkaline  rnethylene-blue  solution.  They  do 
not  produce  acid  by  the  fermentation  of  glucose,  as 
do  all  known  virulent  and  many  non-virulent  diph- 
theria bacilli;  therefore,  there  is  no  increase  in  acidity 
in  the  bouillon  in  which  they  are  grown  during  the 
first  twenty-four  hours  from  the  fermentation  of  the 
meat  sugar  regularly  present.  They  are  found  in  vary- 
ing abundance  in  different  localities  in  about  1  per 
cent,  of  the  normal  throat  and  nasal  secretions,  in  New 
York  City,  and  seem  to  have  now  at  least  no  con- 
nection with  diphtheria;  whether  they  were  originally 
derived  from  diphtheria  bacillus  is  doubtful;  they  cer- 
tainly seem  to  have  no  connection  with  it  now.  They 
never  produce  diphtheria  toxin,  and  to  them  properly 
has  been  applied  the  name  pseudodiphtheria  bacilli.  In 
bouillon  they  grow,  as  a  rule,  less  luxuriantly  than  the 
diphtheria  bacilli.  Some  of  the  varieties  of  the  psewdo- 
diphtheria  bacilli  are  as  long  as  the  shorter  forms  of 
the  virulent  bacilli.  When  these  are  found  in  cultures 


352  BACTERIOLOGY. 

from  cases  of  suspected  diphtheria  they  may  lead  to  an 
incorrect  diagnosis.  The  Neisser  staining  method  is  of 
value  here,  but,  unfortunately,  the  absence  of  the  stained 
bodies  is  not  a  sufficient  ground  to  exclude  the  possi- 
bility of  their  being  true  diphtheria  bacilli.  There  are 
also  some  varieties  which  resemble  the  short  pseudo- 
bacilli  in  form  and  staining,  but  which  produce  acid  in 
glucose  bouillon.  These  bacilli  are  found  occasionally 
in  all  countries  where  search  has  been  made  for  them. 
It  may  be  added  here  that  no  facts  have  come  to  light 
which  indicate  that  bacilli  which  do  not  produce  diph- 
theria toxin  in  animals  ever  produce  it  in  man.  It 
must  also  be  borne  in  mind,  however,  that  such  proof 
is  necessarily  very  difficult  to  obtain. 

Mixed  Infection  in  Diphtheria.  Virulent  diphtheria 
bacilli,  however,  are  not  the  only  bacteria  present  in 
human  diphtheria.  Various  cocci,  more  particularly 
streptococci,  staphylococci,  and  pneumococci,  are  almost 
always  found  associated  with  Loffler  bacilli  in  diph- 
theria, playing  an  important  part  in  the  disease  and 
leading  often  to  serious  complications  (sepsis  and  bron- 
chopneumonia).  Indeed,  the  prognosis  in  a  case  of 
diphtheria  is  now  judged  to  be  graver,  other  things 
being  equal,  according  to  the  degree  in  which  other 
pathogenic  bacteria  influence  the  course  of  the  disease. 
These  cases  of  so-called  mixed  infection  in  diphtheria 
have  within  recent  years  attracted  considerable  atten- 
tion, and  have  been  the  subject  of  a  number  of  animal 
experiments.  Though  the  results  of  these  investiga- 
tions so  far  have  been  somewhat  indefinite,  they  would 
seem  to  iudicate  that  when  other  bacteria  are  associated 
with  the  diphtheria  bacilli  they  mutually  assist  one 
another  in  their  attacks  upon  the  mucous  membrane, 


DIPHTHERIA  BACILLUS.  353 

the  streptococcus  being  particularly  active  in  this 
respect,  often  opening  the  way  for  the  invasion  of  the 
Loffler  bacillus  into  the  deeper  tissues  or  supplying 
needed  conditions  for  the  development  of  its  toxin. 
Thus  diphtheria  is  not  always  a  primary,  but  often  a 
secondary  disease,  following  some  other  infection,  as 
measles  or  scarlet  fever.  In  most  fatal  cases  of  bron- 
chopneumonia  following  laryngeal  diphtheria  we  find 
not  only  abundant  pneumococci  or  streptococci  in  the 
inflamed  lung  areas,  but  also  in  the  blood  and  tissues 
of  the  organs.  As  these  septic  infections  due  to  the 
pyogenic  cocci  are  in  no  way  influenced  by  the  diph- 
theria antitoxin,  they  frequently  are  the  cause  of  the 
fatal  termination.  Other  bacteria  cause  putrefactive 
changes  in  the  exudate,  producing  alterations  in  color 
and  offensive  odors. 

Pseudomembranous  Exudative  Inflammations  Due  to 
Bacteria  other  than  the  Diphtheria  Bacilli.  The  diph- 
theria bacillus,  though  the  most  usual,  is  not  the  only 
micro-organism  that  is  capable  of  producing  pseudo- 
membranous  inflammations.  There  are  numerous  bac- 
teria present  almost  constantly  in  the  throat  secretions, 
which,  under  certain  conditions,  can  (ause  local  lesions 
very  similar  to  those  in  the  less  marked  cases  of  true 
diphtheria.  The  streptococcus  and  pneumococcus  are 
the  two  forms  most  frequently  found  in  these  cases, 
but  there  are  also  others  which,  under  suitable  condi- 
tions, take  an  active  part  in  producing  this  form  of 
inflammation.  Some  of  these  bacteria  do  not  develop 
on  artificial  media,  so  that  we  know  little  of  their 
characteristics.  Among  these  is  a  long  slender  bacillus 
which  is  occasionally  found  in  great  abundance  in  the 
middle  layers  of  pseudomembranes  when  the  diphtheria 

23 


354  BACTERIOLOGY. 

bacillus  is  absent.  This,  or  one  similar  to  it,  has  been 
described  by  Vincent.1  It  does  not  grow  on  artificial 
media  and  is  not  pathogenic  in  animals.  From  its 
presence  in  the  false  membrane  of  a  number  of  cases, 
it  is  believed  to  have  some  causal  relation  to  them. 

These  cases  show  most  of  the  local  appearances  of 
true  diphtheria,  the  superficial  necrosis  of  the  epithe- 
lium, the  membrane  of  the  glandular  swellings.  The 
pseudomembranes  may  persist  for  from  one  to  two 
weeks,  or  even,  in  exceptional  cases,  longer.  This 
bacillus  is  apparently  frequently  present  in  the  normal 
throat,  and  is  probably  only  able  under  certain  favor- 
ing conditions,  such  as  syphilis,  to  produce  lesions. 
Nerve  degeneration  and  paralysis  do  not  follow  an 
attack. 

The  pseudomembranous  angina  accompanying  scarlet 
fever,  and  to  a  less  extent  other  diseases,  may  not 
show  the  presence  of  diphtheria  bacilli,  but  only  the 
pyogenic  cocci,  especially  streptococci,  or,  more  rarely, 
some  varieties  of  little  known  bacilli.  The  deposit 
covering  the  inflamed  tissues  in  these  non-specific  cases 
is,  it  is  true,  usually  but  not  always,  rather  an  exudate 
than  a  true  pseudomembrane.  The  majority  of  these 
cases,  however,  are  mild  affections,  being  only  of  im- 
portance in  adding  to  the  severity  of  the  disease  which 
they  complicate.  An  exception  should  be  made  when 
the  larynx  is  affected,  as  here  the  lungs  are  often  sec- 
ondarily involved.  The  bacteria  which  occur  in  false 
diphtheria  are  streptococci,  staphylococci,  diplococci, 
and  sometimes  pseudodiphtheria  bacilli  or  bacilli  which 
are  morphologically  and  culturally  distinct  from  the 

i  Annales  de  1'Institut  Pasteur,  August,  1899. 


DIPHTHERIA  BACILLUS.  355 

Loffler  bacilli.  These  will  be  referred  to  further  under 
their  respective  organisms. 

The  Transmission  of  Diphtheria.  The  possibility  of 
the  transmission  of  diphtheria  from  animals  to  man 
cannot  be  disputed,  for  cats  and  many  animals  can  be 
infected,  but  there  are  no  authentic  cases  of  such 
transmission  on  record.  So-called  diphtheritic  disease 
in  animals  and  birds  is  usually  due  to  other  micro- 
organisms than  the  diphtheria  bacilli.  Diphtheritic 
infection,  however,  can  generally  be  traced,  directly  or 
indirectly,  to  its  source;  though  there  are  undoubtedly 
some  cases  of  diphtheria  in  which  we  cannot  determine 
the  source  of  the  infection,  for  we  have  no  reason  to 
believe  that  diphtheria  is  ever  spontaneous. 

Let  us  consider  some  of  the  means  by  which  the  dis- 
ease may  be  communicated.  In  actual  experiment  the 
bacilli  have  been  observed  to  remain  virulent  in  bits 
of  dried  membrane  by  Loffler  for  fourteen  weeks, 
by  us  for  seventeen  weeks,  and  by  Roux  and  Yersin 
for  twenty  weeks.  Dried  on  silk  threads  Abel  reports 
that  they  may  sometimes  live  one  hundred  and  seventy- 
two  days,  and  upon  a  child's  plaything  which  had  been 
kept  in  a  dark  place  they  lived  for  five  months.  The 
virulent  bacilli  have  been  found  on  soiled  bedding  or 
clothing  of  a  diphtheria  patient,  on  drin king-cups, 
shoes,  hair,  slate-pencils,  etc.  Beside  these  sources 
of  infection  by  which  the  disease  may  be  indirectly 
transmitted,  virulent  bacilli  may  be  directly  received 
from  the  pseudo membrane,  exudate,  or  discharges  of 
diphtheria  patients;  from  the  secretions  of  the  nose 
and  throat  of  convalescent  cases  of  diphtheria  in  which 
the  virulent  bacilli  persist;  and  from  the  healthy 
throats  of  individuals  who  acquired  the  bacilli  from 


356  BACTERIOLOGY. 

being  in  contact  with  others  having  virulent  germs  on 
their  persons  or  clothing.  In  such  cases  the  bacilli 
may  sometimes  live  and  develop  for  days  or  weeks  in 
the  throat  without  causing  any  lesion.  When  we  con- 
sider that  it  is  only  the  severe  types  of  diphtheria  that 
remain  isolated  during  their  actual  illness,  the  wonder 
is  not  that  so  many,  but  that  so  few,  persons  contract 
the  disease.  It  indicates  that  very  frequently  virulent 
bacilli  are  received  into  the  mouth,  and  then  either  find 
no  conditions  there  suitable  for  their  growth  or  are 
swept  away  by  food  or  drink  before  they  could  effect 
a  lodgement. 

Susceptibility  to  and  Immunity  against  Diphtheria. 
An  individual  susceptibility,  both  general  and  local,  to 
diphtheria,  as  in  all  infectious  diseases,  is  necessary  to 
contract  this  disease.  Moreover,  the  diphtheria  poison 
does  not  produce  the  same  effect  on  the  mucous  mem- 
branes of  all  persons.  Age  has  long  been  recognized 
to  be  an  important  factor  in  diphtheria.  Children 
within  the  first  six  months  of  life. are  but  little  sus- 
ceptible, the  greatest  degree  of  susceptibility  being 
between  the  third  and  the  tenth  year,  while  adults 
are  almost  immune.  An  inherited  susceptibility  or 
"family  predisposition"  to  the  disease  has  also  been 
observed. 

Long  before  the  discovery  of  the  Klebs-Loffler 
bacillus  it  was  a  well  known  fact  that  two  attacks  of 
diphtheria  seldom  occurred  in  the  same  individual  within 
short  periods  of  time,  and  none  of  us  would  fear  to 
leave  a  convalescent  case  in  the  same  room  with  one  still 
suffering  from  the  disease.  To  what  this  natural  suscep- 
tibility or  immunity  is  due  is  still  only  partially  under- 
stood; when  we  remember,  however,  that  simply  a  slight 


DIPHTHERIA  BACILLUS.  357 

increase  in  the  acidity  or  alkalinity  of  the  bouillon  in 
which  the  diphtheria  bacilli  are  producing  their  toxin 
will  prevent  further  production,  it  is  easy  to  imagine  that 
many  changes  in  the  throat  secretion  or  in  its  mucous 
membrane  may  prevent  the  development  of  the  bacil- 
lus or  of  the  production  by  the  bacillus  of  its  toxin, 
and,  therefore,  of  its  disease-producing  power.  But, 
as  the  result  of  animal  experiments,  it  is  now  known 
that  an  artificial  immunity  against  diphtheria  can  be 
produced,  at  least  for  a  considerable  length  of  time, 
by  the  development  of  substances  directly  antidotal  to 
the  diphtheria  toxin.  By  the  inoculation  of  virulent  or 
somewhat  attenuated  cultures  or  of  diphtheria  toxin, 
Fraenkel,  Behring,  Wernicke,  Aronson,  Roux,  and 
since  then  many  others,  have  succeeded  in  immunizing 
animals;  but  the  most  important  and  valuable  results 
are  those  which  have  been  obtained  by  Behring,  in 
conjunction  with  others,  who  showed  that  the  blood 
of  immune  animals  contains  a  substance  which  neutial- 
izes  the  diphtheria  toxin.  The  blood-serum  of  persons 
who  have  recovered  from  diphtheria  has  been  found 
also  to  possess  this  protective  property,  which  it  acquires 
about  a  week  after  the  beginning  of  the  disease,  and  loses 
again  in  a  few  weeks  or  months.  Moreover,  the  blood- 
serum  of  many  individuals,  usually  adults,  who  have 
never  had  diphtheria  often  has  a  slight  general  anti- 
toxic property. 

Antitoxic  Serum.  The  knowledge  derived  from  these 
remarkable  investigations  into  the  protective  powers  of 
the  blood-serum  of  immunized  animals  has  been  em- 
ployed with  the  most  brilliant  results  for  the  prevention 
and  early  treatment  of  diphtheria  in  man.  The  dis- 
covery of  the  method  of  the  production  of  antitoxic 


358  BACTERIOLOGY. 

serum  or  antitoxin  in  animals,  and  its  practical  appli- 
cation to  the  treatment  and  cure  of  diphtheria,  has 
been  shared  by  many  experimenters,  at  first  chiefly 
in  Germany  and  France,  and  later  in  this  country. 
Among  those  whose  labors  in  this  direction  have  ren- 
dered them  most  worthy  of  mention  are  Behring, 
Ehrlich,  Boer,  Kossel,  and  Aronson  in  Germany; 
Roux,  Martin,  and  Chaillou  in  France. 

Results  of  the  Antitoxin  Treatment  of  Diphtheria. 
Though  the  results  of  the  antitoxin  treatment  of 
diphtheria  belong  properly  to  the  province  of  serum- 
therapy  rather  than  to  bacteriology,  in  view  of  the 
great  practical  importance  of  the  subject  it  may  not  be 
amiss  to  quote  here  the  conclusions  arrived  at  by  Biggs 
and  Guerard  after  a  review  of  all  the  statistics  and 
opinions  published  since  the  beginning  of  the  antitoxin 
treatment  in  1892  : 

"  It  matters  not  from  what  point  of  view  the  sub- 
ject is  regarded,  if  the  evidence  now  at  hand  is  properly 
weighed,  but  one  conclusion  is  or  can  be  reached — 
whether  we  consider  the  percentage  of  mortality  from 
diphtheria  and  croup  in  cities  as  a  whole,  or  in  hospi- 
tals, or  in  private  practice;  or  whether  we  take  the 
absolute  mortality  for  all  the  cities  of  Germany  whose 
population  is  over  15,000,  and  all  the  cities  of  France 
whose  population  is  over  20,000;  or  the  absolute  mor- 
tality for  New  York  City,  or  for  the  great  hospitals  in 
France,  Germany,  and  Austria;  or  whether  we  con- 
sider only  the  most  fatal  cases  of  diphtheria,  the  laryn- 
geal  and  operative  cases;  or  whether  we  study  the  ques- 
tion with  relation  to  the  day  of  the  disease  on  which 
treatment  is  commenced,  or  the  age  of  the  patient 
treated;  it  matters  not  how  the  subject  is  regarded  or 


DIPHTHERIA  BACILLUS.  359 

how  it  is  turned  for  the  purpose  of  comparison  with 
previous  results,  the  conclusion  reached  is  always  the 
same — namely,  there  has  been  an  average  reduction  of 
mortality  from  the  use  of  antitoxin  in  the  treatment  of 
diphtheria  of  not  less  than  50  per  cent.,  and  under  the 
most  favorable  conditions  a  reduction  to  one-quarter, 
or  even  less,  of  the  previous  death-rate.  This  has 
occurred  not  in  one  city  at  one  particular  time,  but  in 
many  cities,  in  different  countries,  at  different  seasons 
of  the  year,  and  always  in  conjunction  with  the  intro- 
duction of  antitoxin  serum  and  proportionate  to  the 
extent  of  its  use.7' 

The  Production  of  Diphtheria  Antitoxin  for  Therapeutic 
Purposes.  As  a  result  of  the  work  of  years  in  the 
laboratories  of  the  Health  Department  of  New  York 
City,  the  following  may  be  laid  down  as  a  practical 
method  : 

The  strongest  diphtheria  toxin  possible  should  be 
obtained  by  taking  a  very  virulent  culture  and  grow- 
ing it  under  the  conditions  described  on  page  345. 
The  culture,  after  a  week's  growth,  is  removed,  and 
having  been  tested  for  purity  by  microscopical  and 
culture  tests  is  rendered  sterile  by  the  addition  of  10 
per  cent,  of  a  5  per  cent,  solution  of  carbolic  acid. 
On  the  following  day  the  sterile  culture  is  filtered 
through  ordinary  sterile,  filter-paper  and  stored  in 
full  bottles  in  a  cold  place  until  needed.  Its  strength 
is  then  tested  by  giving  a  series  of  guinea-pigs  care- 
fully measured  amounts.  Less  than  0.01  c.c.,  when 
injected  hypodermatically,  should  kill  a  250-gramme 
guinea-pig. 

The  horses  used  should  be  young,  vigorous,  of  fair 
size,  and  absolutely  healthy.  Vicious  habits,  such  as 


360  BACTERIOLOGY. 

kicking,  etc.,  make  no  difference,  of  course,  except  to 
those  who  handle  the  animals.  A  number  of  such 
horses  are  severally  injected  with  an  amount  of  toxin 
sufficient  to  kill  five  thousand  guinea-pigs  of  250 
grammes'  weight  (about  20  c.c.  of  strong  toxin). 
After  from  three  to  five  days,  so  soon  as  the  fever 
reaction  has  subsided,  a  second  subcutaneous  injection 
of  a  slightly  larger  dose  is  given.  With  the  first  three 
injections  of  toxin  10,000  units  of  antitoxin  are  given. 
If  antitoxin  is  not  mixed  with  the  first  doses  of  toxin 
only  one-tenth  of  the  doses  advised  is  to  be  given. 
At  intervals  of  from  five  to  eight  days  increasing  injec- 
tions of  pure  toxin  are  made,  until  at  the  end  of  two 
months  from  ten  to  twenty  times  the  original  amount 
is  given.  There  is  absolutely  no  way  of  judging  which 
horses  will  produce  the  highest  grades  of  antitoxin. 
Very  roughly,  those  horses  which  are  extremely  sensi- 
tive and  those  which  react  hardly  at  all  are  the  poorest, 
but  even  here  there  are  exceptions.  The  only  way, 
therefore,  is  at  the  end  of  six  weeks  or  two  months  to 
bleed  the  horses  and  test  their  serum.  If  only  high- 
grade  serum  is  wanted  all  horses  that  give  less  than 
150  units  per  c.c.  are  discarded.  If  moderate  grades 
only  are  desired,  all  that  yield  100  units  may  be 
retained.  The  retained  horses  receive  steadily  in- 
creasing doses,  the  rapidity  of  the  increase  and  the 
interval  of  time  between  the  doses  (three  days  to  one 
week)  depending  somewhat  on  the  reaction  following 
the  injection,  an  elevation  of  temperature  of  more  than 
3°  F.  being  undesirable.  At  the  end  of  three  months 
the  antitoxic  serum  of  all  the  horses  should  contain 
over  300  units,  and  in  about  10  per  cent,  as  much  as 
800  units  in  each  cubic  centimetre.  Very  few  horses 


DIPHTHERIA  BACILLUS.  361 

ever  give  above  1000  units,  and  none  so  far  has  given 
as  much  as  2000  units  per  c.c.  The  very  best  horses 
continue  to  furnish  blood  containing  the  maximum 
amount  of  antitoxin  for  several  months,  and  then,  in 
spite  of  increasing  injections  of  toxin,  begin  to  furnish 
blood  of  gradually  decreasing  strength.  If  every  nine 
months  an  interval  of  three  months'  freedom  from 
inoculations  is  given,  the  best  horses  furnish  high- 
grade  serum  during  their  periods  of  treatment  for  from 
two  to  four  years. 

In  order  to  obtain  the  serum  the  blood  is  withdrawn 
from  the  jugular  vein  by  means  of  a  sharp- pointed 
canula,  which  is  plunged  through  the  vein  wall,  a  slit 
having  been  made  in  the  skin.  The  blood  is  carried 
by  a  sterile  rubber  tube  into  large  Erlenmeyer  flasks 
and  allowed  to  clot,  the  flasks,  however,  being  placed 
in  a  slanting  position  before  clotting  has  commenced. 
The  serum  is  drawn  off  after  four  days  by  means  of  sterile 
glass  and  rubber  tubing,  and  is  stored  in  large  flasks. 
From  this,  as  needed,  small  phials  are  filled.  The 
phials  and  their  stoppers,  as  indeed  all  the  utensils  used 
for  holding  the  serum,  must  be  absolutely  sterile,  and 
every  possible  precaution  must  be  taken  to  avoid 
contamination  of  the  serum.  An  antiseptic  may  be 
added  to  the  serum  as  a  preservative,  but  it  is  not 
necessary  and  probably  inadvisable,  except  when  the 
serum  is  to  be  sent  to  great  distances,  where  it  cannot 
be  kept  under  supervision. 

Kept  from  access  of  air  and  light  and  in  a  cold  place 
it  is  fairly  stable,  deteriorating  not  more  than  40  per 
cent.,  and  often  much  less,  within  a  year.  Diphtheria 
antitoxin,  when  stored  in  phials  and  kept  under  the 
above  conditions,  contains  within  10  per  cent,  of  its 


362  BACTEEIOLOOY. 

original  strength  for  at  least  two  months;  after  that  it 
can  be  used  by  allowing  for  a  maximum  deterioration 
of  10  per  cent,  for  each  month.  The  antitoxin  in  old 
serum  is  just  the  same  as  in  that  freshly-bottled,  only 
there  is  less  of  it. 

The  nature  of  diphtheria  antitoxin  has  until  recently 
been  known  almost  wholly  from  its  physiological  prop- 
erties. Recently  experiments  have  seemed  to  show  that 
it  was  either  closely  bound  to  the  globulins  or  was  itself 
a  globulin.  Mr.  J.  P.  Atkinson,  assistant  chemist  in 
the  laboratory,  has  kindly  permitted  me  to  state  the 
results  of  his  investigations,  which  will  soon  appear  in 
the  Journal  of  Experimental  Medicine.  He  found  that 
antitoxic  and  normal  horse-serum  react  similarly  toward 
M'gSO4,  in  that  the  globulin  is  precipitated  completely 
from  the  other  constituents  of  the  serum.  In  the  case 
of  antitoxic  serum  the  globulin  precipitate  carries  with 
it  all  of  the  antitoxic  power  of  the  serum,  leaving  the 
filtrate  without  any  neutralizing  power  against  the  diph- 
theria toxin.  When  watery  solutions  of  this  globulin 
are  saturated  with  NaCl  a  precipitate  occurs.  When 
the  solution  is  heated  a  series  of  further  precipitates  take 
place,  as  follows  :  Cloudiness  appears  at  40°,  49°,  57°, 
and  67°  C. ;  complete  precipitate  occurs  at  45°,  54°, 
62°,  and  72°  C.  Each  of  these  precipitates  has  anti- 
toxic properties,  and  the  total  quantities  contain  all  the 
original  antitoxin  except  some  5  per  cent.,  which  is  evi- 
dently destroyed  by  the  higher  temperatures  required 
for  the  last  two  precipitates.  After  the  last  precipitate 
the  solution  is  free  of  globulin  and  also  of  all  antitoxic 
properties. 

A  further  fact  developed  by  Atkinson  is  that  the 
globulins  increase  markedly  in  the  serum  of  horses  as 


DIPHTHERIA  BACILLUS.  363 

the  antitoxin  strength  increases.  It  seems,  therefore, 
from  the  above  facts  that  diphtheria  antitoxin  has  the 
characteristics  of  the  globulins.  Whether  it  is  a  union 
of  diphtheria  toxin  and  globulin,  or  an  increase  of  cer- 
tain globulin-like  substances  through  the  stimulation  of 
the  toxin,  we  have  as  yet  no  facts  to  tell  us.  Antitoxin 
is  destroyed  by  prolonged  moderate  heat  (60°  C.)  and 
by  short  exposure  to  higher  temperatures  (95°  to  100° 
C.).  It  is  much  less  sensitive  than  diphtheria  toxin. 

Diphtheria  antitoxin  has  the  power  of  neutralizing 
diphtheria  toxin,  so  that  when  a  certain  amount  is  in- 
jected into  an  animal  before  or  together  with  the  toxin 
it  overcomes  its  poisonous  action.  As  already  stated, 
there  is  a  great  difference  of  opinion  as  to  whether  anti- 
toxin acts  by  direct  chemical  neutralization  of  the  toxin 
or  indirectly  on  the  cells.  The  facts  in  favor  of  a  direct 
action  of  antitoxins  upon  their  corresponding  toxins 
have  recently  been  briefly  summarized  by  Cobbett  as 
follows  : 

1.  Certain   reactions    have    been   observed   to   take 
place  between  these  substances  outside  the  animal  body 
(venom,  ricln,  crotin,  tetanus  toxin,  diphtheria  toxin, 
and  their  corresponding  antitoxins). 

2.  Various  attempts  to  separate  the  toxins  and  anti- 
toxins from  neutral  mixtures  have  been  failures.     Par- 
tial successes  have,   at  least  in   some  instances,  been 
shown  to  depend  upon  the  fact  that  insufficient  time  for 
their  complete  union  was  allowed,  separation  being  no 
longer  possible  if  this  were  granted. 

3.  The  accuracy  of  the  titration  of  toxins  and  anti- 
toxins to  within  1  per  cent,  of  error. 

4.  The  fact  that  to  save  an  animal  from  1000  fatal 
doses  of  diphtheria  toxin  requires  little  more  than  a 


364  BACTERIOLOGY. 

hundred  times  as  much  antitoxin  as  is  required  for  ten 
fatal  doses,  the  resistance  of  the  animal  it-elf  account- 
ing for  the  difference. 

5.  The  fact  that  the  potency  of  antitoxin  is  greatly 
increased  if  it  is  allowed  to  come  in  contact  with  the 
toxin  outside  the  animal  body;  and  is  increased  still 
further  if  allowed  to  remain  for  sufficient  time  in  con- 
tact with  the  toxin  at  a  suitable  temperature. 

On  the  other  hand,  the  conclusions  which  Buchner 
and  Roux  drew  from  their  experiments  have  been  shown 
to  have  been  based  on  a  misconception,  for  they  ignored 
the  capacity  of  an  animal  to  deal  with  a  certain  minimal 
quantity  of  poison,  and,  consequently,  made  no  distinc- 
tion between  a  physiologically  neutral  and  a  completely 
neutral  mixture. 

The  facts  now  known,  therefore,  indicate  rather 
strongly  that  the  antitoxins  of  tetanus  and  diphtheria, 
of  snake-poison,  of  ricin,  etc.,  enter  into  direct  chem- 
ical combination  with  their  respective  toxins — a  com- 
bination which  is,  perhaps,  not  exactly  comparable  to 
that  of  an  acid  with  an  alkali;  for,  as  we  have  seen, 
it  is  a  much  slower  one,  but  one  which  possibly — as 
Ehrlich  has  suggested — more  closely  resembles  the  for- 
mation of  a  double  salt.  Some  facts  seem  to  indicate 
that  the  antitoxin  has  a  stronger  affinity  for  toxin  than 
the  toxin  has  for  the  cells.  Many  points,  however,  are 
still  far  from  clear  as  to  the  manner  in  which  both 
toxins  and  antitoxins  act. 

The  Testing  of  Antitoxin.  This  power,  possessed 
by  a  definite  quantity  of  antitoxin  to  neutralize  a  cer- 
tain amount  of  toxin,  is  utilized  in  testing  antitoxin. 
Guinea-pigs  of  about  250  grammes'  weight  are  sulicu- 
taneously  injected  with  one  hundred  or  with  ten  fatal 


DIPHTHERIA  BACILLUS.  365 

doses  of  toxin  which  have  been  previously  mixed  with 
an  amount  of  antitoxin  believed  to  be  sufficient  to  pro- 
tect from  the  toxin.  If  the  guinea-pig  lives  four  days, 
but  dies  soon  after,  the  amount  of  antitoxin  added  to 
the  toxin  was  just  1  or  0.1  unit,  according  as  one  hun- 
dred or  ten  fatal  doses  were  employed.  If  the  guinea- 
pig  dies  earlier,  less  than  1  unit  was  added. 

The  Use  of  Antitoxin  in  Treatment  and  Immuniza- 
tion. The  antitoxin  in  the  higher  grades  is  iden- 
tical with  that  in  the  lower  grades  ;  there  is  simply 
more  of  it  in  each  drop  of  the  serum.  In  treatment, 
however,  for  the  same  amount  of  antitoxin  we  have  to 
inject  less  blood-serum  with  the  higher  grades,  and, 
therefore,  have  somewhat  less  danger  of  rashes  and 
other  deleterious  results.  With  concentrated  globulin 
solutions  we  may  hope  still  further  to  avoid  all  dis- 
agreeable effects  (see  page  362).  The  amount  of  anti- 
toxin required  for  immunization  is  200  units  for  an 
infant,  500  for  an  adult,  and  proportionately  for  those 
between  these  extremes.  After  the  observation  of  the 
use  of  antitoxin  in  the  immunization  of  several  thousand 
cases,  I  have  absolute  belief  in  its  power  to  prevent 
an  outbreak  of  diphtheria  for  at  least  two  weeks,  and 
also  of  its  harmlessness  in  the  small  doses  required.  If 
it  is  desired  to  prolong  the  immunity  the  antitoxin  in- 
jection is  repeated  every  two  weeks.  For  treatment, 
mild  cases  should  be  given  1500  units,  moderate  cases 
2000  units,  and  severe  cases  3000  units.  Where  no 
improvement  follows  in  twelve  hours  the  dose  should 
be  repeated.  Antitoxin  is  useless  when  given  by  the 
mouth,  as  very  little  of  it  is  absorbed. 

No  deleterious  effects  are  to  be  feared  except  a  rash, 
with  some  rise  of  temperature,  in  about  20  per  cent,  of 


366  BACTERIOLOGY. 

the  cases.  With  the  serum  from  some  horses  the  rashes 
are  very  infrequent,  while  with  that  from  others  they 
occur  more  often.  The  same  horse  will  at  one  time 
furnish  a  serum  which  produces  no  rashes  and  at  an- 
other one  which  gives  a  great  number.  No  way  has 
yet  been  found  to  eliminate  them  entirely.  Filtering 
and  moderate  heating  produce  little  effect.  Standing 
for  some  months  causes  a  precipitate  to  occur,  and  the 
clear  serum  seems  somewhat  less  liable  to  produce 
rashes  than  when  it  was  fresh. 

The  Persistence  of  Antitoxin  in  the  Blood.  When  in- 
jections of  toxin  are  stopped  in  a  horse  the  antitoxin  is 
slowly  eliminated,  so  that  there  is  a  loss  of  about  20 
per  cent,  a  week.  In  from  three  to  five  months  all 
appreciable  antitoxin  has  been  eliminated.  Immunity 
in  human  beings  lasts  from  two  to  six  weeks  after  an 
injection  of  500  units  of  antitoxin. 

Technical  Points  upon  the  Testing  of  Diphtheria  Anti- 
toxin and  the  Relations  between  the  Toxicity  and  Neu- 
tralizing Value  of  Diphtheria  Toxin.  Until  within  a 
fairly  recent  time  the  filtered  or  sterilized  bouillon  in 
which  the  diphtheria  bacillus  had  grown  and  produced 
its  "  toxin"  was  supposed  to  require  for  its  neutraliza- 
tion an  amount  of  antitoxin  directly  proportional  to  its 
toxicity  as  tested  in  guinea-pigs.  Thus,  if  from  one 
bouillon  culture  ten  fatal  doses  of  "  toxin"  were  re- 
quired to  neutralize  a  certain  quantity  of  antitoxin,  it 
was  believed  that  ten  fatal  doses  from  every  culture, 
without  regard  to  the  way  in  which  it  had  been  pro- 
duced or  preserved,  would  also  neutralize  the  same 
amount  of  antitoxin.  Upon  this  belief  was  founded 
the  Behring-Ehrlich  definition  of  an  antitoxin  unit. 

The  results  of  tests  by  different  experimenters  with 


DIPHTHERIA  BACILLUS. 


367 


the  same  antitoxic  serum,  but  with  different  diphtheria 
toxins,  proved  this  opinion  to  be  incorrect.  Ehrlich1 
deserves  the  credit  for  first  clearly  perceiving  and 
publishing  this.  He  obtained  from  various  sources 
twelve  toxins  and  compared  their  neutralizing  value 
upon  antitoxin  ;  these  tests  gave  most  interesting  and 
important  information.  The  results  in  six  toxins, 
which  are  representative  of  the  twelve,  are  as  shown 
in  the  following  table : 


o> 

a 

°i 

'*""'  '^   O  L^   ^     j  /^ 

JfJI*. 

£ 

a 

|| 

2^2|g? 

l§"  «?1 

a 

.2  ^ 

laSf^  +  ^"a^lsSj  L,-Ln 

'oJ3 

si 

£z; 

*sl| 

-§'0  cS 

9(-1^^T3*-^    S^^.^3  >>.5  o3  o 

^Sobcg:,  isS^^Ss?  =fatal 
a^^-a-as  'gS'S-g   sc-a  doses- 

^Ou^gP^3     fOO^^Jr^U3&^ 

Data  upon  "  toxin" 
specimen  given  by 
Ehrlich. 

_.  J3       1       >       O> 
SS         9-3  fl 

^w-sSSfl  :_S5'S5'3^ 

^-tDPCfl>-^H    *3  R  T5  B      »3   o; 

p^J       .SJ-2 

1  JB  &3  S  y  ^  8  2  S     3_! 

E-i  ° 

^=2^ 

|-§2a^s 

^=^^oa. 

2 

0.03 

42 

32 

10 

Preserved  two  years. 

4 

0.009 

39.4 

33.4 

6 

Old,  deteriorated  from 

0.003  to  0.009. 

7 

0.0165 

76.3 

54.4 

22 

Fresh  toxin,  preserved 

with  tricresol. 

9 

0.039 

123 

108 

15 

A  number  of  fresh  cul- 

tures grown  at  37°  C. 
four  and  eight  days. 

10 

0.001 

29.2 

27.5 

1.7 

Precipitated   from 
greatly  deteriorated 

"toxin." 

12 

0.0025 

100 

50 

50 

Tested  immediately 
after  its  withdrawal. 

From  the  facts  set  forth  in  the  tables,  Ehrlich  has 
derived  interesting  theories,  which,  if  true,  would  add 
greatly  to  our  knowledge  of  toxins,  and  would  also  have 
a  very  direct  influence  upon  the  present  methods  of 
standardizing  antitoxin.  He  believes  that  the  diphtheria 
bacilli  in  their  growth  produce  a  toxin  which,  so  long  as 


1  "  Die  Wertbemessung  des  Diphtherieheilserums  und  deren  theoretische 
Grundlagen,"  Klinisches  Jahrbuch,  1897. 


368  BA  CTERIOL  OGY. 

it  remains  chemically  unaltered,  has  a  definite  poisonous 
strength  with  a  definite  value  in  neutralizing  antitoxin. 
This  neutralization  he  believes  to  be  a  chemical  union, 
in  which  two  hundred  fatal  doses  of  toxin  for  a  250 
grammes'  weight  guinea-pig  combine  with  one  unit  of 
antitoxin.  The  toxin  is,  however,  an  unstable  com- 
pound, and  begins  to  change  almost  immediately  into 
substances  which  are  not,  at  least  acutely,  poisonous, 
but  which  retain  their  full  power  to  neutralize  anti- 
toxin. These  substances,  according  to  Ehrlich,  fall 
into  three  groups.  The  first  has  more  affinity  for 
combining  with  the  antitoxin  than  the  toxin  itself 
(protoxoids).  The  second  has  the  same  affinity  (syn- 
toxoids).  The  third  has  less  affinity  (epitoxoids). 

According  to  him,  if  a  mixture  of  toxoids  and  toxin 
is  added  to  antitoxin,  the  protoxoids  first  combine  with 
the  antitoxin,  then  the  syntoxoids  and  the  toxin  com- 
bine in  equal  proportions,  so  long  as  the  supply  lasts, 
with  the  amount  of  antitoxin  remaining,  or,  if  there 
is  a  surplus,  with  enough  to  satisfy  them;  finally,  if 
any  antitoxin  remains,  the  epitoxoids  unite  with  it. 

If  to  a  mixture  in  which  all  three  toxoids,  as  well  as 
toxin,  have  united  with  antitoxin,  some  additional  toxic 
culture  bouillon  be  added,  the  new  protoxoids  displace 
first  the  epitoxoids,  and  then,  if  free  protoxoids  remain, 
the  toxin  and  the  syntoxoids  from  their  antitoxin,  and 
thus  liberate  as  well  as  add  free  toxin  to  the  solution. 

Ehrlich  gives  an  interesting  theory  to  explain  the  pro- 
duction of  antitoxin  in  the  blood.  This  he  does  upon 
the  supposition  that,  when  absorbed,  the  toxin  combines 
with  a  portion  of  certain  selected  cells,  and  that  this 
portion,  by  its  union  with  toxin,  becomes — at  least 
physiologically — dead.  The  cell  replaces  this  dead 


DIPHTHERIA  BACILLUS.  369 

matter  with  new  and  similar  substance;  after  the  stimuli 
following  several  repeated  losses  and  replacements  of 
this  substance  the  cells  produce  it  in  excess.  This  sub- 
stance, whether  originally  in  the  normal  cell  or  repro- 
duced there,  and  whether  remaining  in  the  cell  or 
thrown  out  into  the  circulation,  is  antitoxin. 

The  above  summary  merely  gives  an  outline  of 
some  of  the  points  in  Ehrlich' s  most  interesting  article. 
To  become  fully  acquainted  with  the  reason  for  his 
theories  the  article  itself  must  be  carefully  read. 

Interest  in  both  his  theoretical  reasoning  and  in  his 
practical  conclusions  led  us  to  subject  both  to  a  series 
of  tests  which  have,  I  believe,  added  some  interesting 
facts  to  those  already  published  by  Ehrlich  as  well  as 
cast  doubts  on  some  of  his  conclusions. 

The  results  of  these  experiments  of  Atkinson  and 
myself *  were  fully  in  accord  with  those  published  by 
Ehrlich  as  to  the  varying  neutralizing  value  of  a 
minimal  fatal  dose  of  "toxin";  they,  however,  also 
indicate  roughly  a  general  law  in  accordance  with  which 
these  changes  occur. 

The  neutralizing  value  of  a  fatal  dose  of  toxin  is  at 
its  lowest  in  the  culture  fluid  when  the  first  consider- 
able amounts  of  toxin  have  been  produced.  After  a 
short  period,  during  which  the  quantity  of  toxin  in  the 
fluid  is  increasing,  the  neutralizing  value  of  the  fatal  dose 
begins  to  increase,  at  first  rapidly,  then  more  slowly. 

While  the  culture  is  still  in  vigorous  growth  and  new 
toxin  is  being  produced,  the  neutralizing  value  of  the 
fatal  dose  fluctuates  somewhat,  but  with  a  generally 
upward  tendency.  After  the  cessation  of  toxin  pro- 

1  Journal  of  Experimental  Medicine,  vol.  Hi.,  No.  4. 
24 


370  BACTERIOLOGY. 

duction  the  neutralizing  value  of  the  fatal  dose  increases 
steadily  until  it  becomes  five  to  ten  times  its  original 
amount. 

In  our  experiments  the  greatest  value  for  L+  was 
126,  the  least  27.  As  at  six  hours  L+  was  only  72 
and  at  twenty-eight  hours  only  91,  we  doubt  whether 
L^  ever  reaches  above  150.1  When  we  seek  to  analyze 
the  above-described  process  we  find  certain  facts  which 
seem  partly  to  explain  it.  Experiments  have  shown 
that  filtered  toxin,  preserved  for  any  length  of  time  in 
conditions  under  which  access  of  air  occurs,  gradually 
loses  in  both  its  toxicity  and  neutralizing  power,  and 
that  it  loses  more  rapidly  in  the  former  property  than 
in  the  latter.  Thus,  while  the  fatal  dose  of  a  toxin  pre- 
served for  one  year  rose  from  0.01  c.c,  to  0.55  c.c.,  it 
lost  only  half  as  much  in  neutralizing  value,  1  unit 
neutralizing  at  first  1  c.c.,  at  the  end  of  the  year  0.25 
c.c.  These  processes  take  place  more  rapidly  at  room- 
temperature  than  in  the  ice-chest,  and  in  the  incubator 
than  in  the  room. 

In  the  fluid  holding  the  living  bacilli  we  have,  there- 
fore, after  the  first  few  hours  of  toxin  formation,  a 
double  process  going  on — one  of  deterioration  in  the 
toxin  already  accumulated,  which  tends  to  increase  the 
neutralizing  value  of  the  fatal  dose;  the  other  of  new 
toxin  formation,  which  probably  tends  to  diminish  the 
neutralizing  value.  The  chemical  changes  produced 
by  the  growth  of  the  bacilli  in  the  bouillon  tend  to  aid 
one  or  the  other  of  these  processes,  and  so  to  make,  from 
hour  to  hour,  slight  changes  in  the  value  of  the  fatal 


1  Li  =  fatal  doses  of  toxin  required  to  kill  a  guinea-pig  in  four  days  after 
having  been  mixed  with  one  unit  of  antitoxin. 
L0  =  fatal  doses  of  toxin  required  to  fully  neutralize  one  unit  of  antitoxin- 


DIPHTHERIA  BACILLUS.  371 

dose.  Later,  with  the  period  of  cessation  of  toxin  pro- 
duction, the  gradual  deterioration  of  the  toxicity  alone 
continues,  and  the  fatal  dose  gradually  and  steadily  in- 
creases in  its  neutralizing  value. 

Ehrlich's  theories  as  to  the  splitting  up  of  "  toxin" 
into  toxoids  having  little  or  no  toxicity,  but  on  the 
average  full  neutralizing  power  for  antitoxin  have 
not,  in  our  opinion,  been  substantiated  by  the  results 
of  these  experiments.  The  difference  between  the 
amount  of  toxin  mixed  with  a  unit  of  antitoxin  which 
causes  the  first  symptoms  and  that  causing  death  upon 
the  fourth  day  would  be,  it  is  true,  explained  by  his 
theory;  but  the  failure  of  this  difference  to  be  greater 
where,  by  his  theories,  epitoxoids  should  be  in  great 
abundance,  prevents  our  acceptance  of  his  views.  The 
fact  of  the  greater  neutralization  value  of  a  fatal  dose 
of  a  deteriorated  toxin  would  be  accounted  for  on  his 
protoxoid  theory.  This,  however,  is  not  proof  of  its 
correctness,  as  other  theories,  such  as  the  production  by 
the  diphtheria  bacillus  of  two  or  more  closely  allied 
toxins,  similar  to  the  allied  alkaloids  produced  by 
plants,  would  equally  account  for  it  if  we  supposed 
the  one  which  had  the  greater  neutralization  value  was 
more  resistant  to  destruction  than  the  other.1  We  only 
advance  this  theory  to  call  attention  to  the  fact  that 
many  theories  can  on  paper  explain  a  process  without 
necessarily  being  thereby  established. 

While  we  do  not  believe,  therefore,  that  he  has 
changed  the  principles  of  testing  antitoxin,  yet  we 
believe  he  has  contributed  greatly  to  uniformity  in 
results  by  calling  attention  to  the  necessity  of  selecting 

1  The  incomplete  precipitation  of  the  diphtheria  toxin  by  MgSO4  makes  it 
probable  that  more  than  one  poison  exists. 


372  BACTERIOLOGY. 

a  suitable  toxin  and  by  employing  and  distributing  an 
antitoxin  as  a  standard  to  test  toxins  by.  In  this  way 
smaller  testing  stations  can  make  their  results  corre- 
spond with  those  of  the  central  station. 

In  spite  of  the  great  variations  in  the  neutralizing 
value  of  a  fatal  dose  in  different  toxins  we  do  not  be- 
lieve there  has  been  any  such  great  difference  in  the 
toxins  used  by  the  different  stations  for  testing  pur- 
poses. Most  laboratories  have  taken  the  culture  fluid 
at  about  the  time  of  its  greatest  toxicity,  and  the 
neutralizing  value  of  a  fatal  dose  of  this  toxin  would 
seldom  vary  more  than  10  per  cent,  above  or  below  the 
standard  now  adopted  in  Germany  by  the  government 
testing  station,  this  latter  being  presumably  as  close  as 
possible  to  that  used  to  establish  the  original  Behring- 
Ehrlich  unit. 

Where  error  has  been  made  it  has  usually  been  by 
taking  too  old  culture  fluids,  which  would  cause  the 
antitoxin  strength  of  samples  tested  to  be  estimated 
below  and  not  above  its  real  value.  Culture  8,  which  is 
used  not  only  by  the  New  York  Board  of  Health  Labor- 
atory but  by  many  other  laboratories  in  the  United 
States  and  Europe,  fortunately  produces  on  the  sixth 
to  eighth  day — the  time  at  which  the  culture  is  usually 
removed — a  toxin  which  grades  Ehrlich's  antitoxin 
within  5  per  cent,  of  the  strength  given  by  him. 

We  believe  that  by  using  such  a  bacillus  we  can, 
after  gaining  a  fuller  knowledge  of  its  characteristics, 
obtain  a  toxin  of  a  known  and  suitable  neutralizing 
value,  and  thus  always  correctly  standardize  an  anti- 
toxic serum.  This  is  certainly  true  for  the  bacillus 
which  we  have  used  for  the  past  four  years.  Mean- 
while, a  fairly  permanent  preparation  of  a  carefully 


DIPHTHERIA  BACILLUS.  373 

tested  antitoxin  is  of  immense  value  in  insuring  a 
uniform,  though  not  necessarily  correct,  standard  among 
the  different  testing  stations  and  in  allowing  of  com- 
parison between  them. 

The  old  definition  of  Behring  and  Ehrlich,  that  an 
antitoxin  unit  contains  the  amount  of  antitoxin  which 
will  protect  the  life  of  a  guinea-pig  from  one  hundred 
fatal  doses  of  toxin,  must  be  modified  so  as  to  be  de- 
fined as  that  amount  of  antitoxin  which  will  neutral- 
ize one  hundred  fatal  doses  of  a  toxin  similar  to  that 
adopted  as  the  standard — namely,  one  having  the  char- 
acteristics of  toxins  in  cultures  at  the  height  of  their 
toxicity. 

The  actual  test  of  an  antitoxin  serum  is,  therefore, 
carried  out  as  follows:  Six  guinea-pigs  are  injected 
with  mixtures  of  toxin  and  antitoxin.  In  each  of  the 
mixtures  there  is  100  times  the  amount  of  a  toxin  such 
as  just  described,  which  will  kill  250  grammes  of 
guinea-pig  on  an  average  in  96  hours.  In  each  of  the 
mixtures  the  amount  of  antitoxin  varies;  for  instance, 
No.  1  would  contain  0.002  c.c.  serum,  No.  2,  0.003 
c.c.,  No.  3,  0.004  c.c.,  No.  4,  0.005  c.c.,  etc.  If,  at 
the  end  of  the  fourth  day,  Nos.  1,  2,  and  3  were  dead, 
and  Nos.  4,  5,  and  6  were  alive,  we  would  consider 
the  serum  to  contain  200  units  of  antitoxin  for  each  c.c. 
When  we  mix  only  ten  fatal  doses  of  toxin  with  one- 
tenth  of  the  amount  of  antitoxin  used  with  100  fatal 
doses  we  usually  consider  that  the  guinea-pig  must  not 
only  live  but  remain  well. 

The  Relation  of  Bacteriology  to  Diagnosis.  I  believe 
that  all  experienced  clinicians  will  agree  that,  when 
left  to  judge  solely  by  the  appearance  and  symptoms 
of  a  case,  there  are  certain  mild  exudative  inflamma- 


374  BACTERIOLOGY. 

tions  of  the  throat  belonging  to  both  diphtheritic  and 
to  non-diphtheritic  inflammations  which  appear  exactly 
alike,  having  apparently  similar  symptoms  and  similar 
duration;  that  it  is  even  possible  to  examine  two  cases, 
knowing  that  one  is  surely  diphtheria,  or  at  least  that 
diphtheria  bacilli  are  present  in  the  exudate,  and  the 
other  surely  is  not,  and  yet  be  unable  to  determine 
clinically  which  is  true  diphtheria  and  which  is  pseudo- 
diphtheria.  It  is  not  meant  to  imply  that  a  case  is 
one  of  true  diphtheria  simply  because  the  diphtheria 
bacilli  are  present,  but  rather  that  the  doubtful  cases 
not  only  have  the  diphtheria  bacilli  in  the  exudate, 
but  are  capable  of  giving  true  characteristic  diphtheria 
to  others,  or  later  develop  it  characteristically  them- 
selves; and  that  those  in  whose  throats  no  diphtheria 
bacilli  exist  can  under  no  condition  give  true  character- 
istic diphtheria  to  others  or  develop  it  themselves  unless 
they  receive  a  new  infection.  It  is,  indeed,  true,  as  a 
rule,  that  cases  presenting  the  appearance  of  ordinary 
follicular  tonsillitis  in  adults  are  not  due  to  the  diph- 
theria bacillus.  It  is  also  true  that  now  and  then  a  case 
having  this  appearance  is  one  of  diphtheria,  and  almost 
every  physician  has  seen  such  cases  from  time  to  time 
in  households  infected  with  diphtheria.  On  the  other 
hand,  in  small  children  mild  diphtheria  very  frequently 
occurs  with  the  semblance  of  rather  severe  ordinary 
follicular  tonsillitis,  due  to  the  pyogenic  cocci,  and  in 
large  cities  where  diphtheria  is  prevalent  all  such  cases 
must  be  watched  as  being  more  or  less  suspicious. 
As  showing  our  doubt  in  our  own  judgment,  I  think 
most  would  feel  that  if  in  any  case  exposure  to  diph- 
theria is  known  to  have  occurred,  even  a  slightly  sus- 
picious sore-throat  would  be  regarded  as  probably  due  to 


DIPHTHERIA  BACILLUS.  375 

the  diphtheria  bacilli.  If,  on  the  other  hand,  no  cases 
of  diphtheria  have  been  known  to  exist  in  the  neigh- 
borhood, even  cases  of  a  more  suspicious  nature  would 
probably  not  be  regarded  as  diphtheria.  • 

Appearances  Characteristic  of  Diphtheria.  The  pres- 
ence of  irregular-shaped  patches  of  adherent  grayish  or 
yellowish-gray  pseudomembrane  on  some  other  por- 
tions than  the  tonsils  is,  as  a  rule,  an  indication  of  the 
activity  of  the  diphtheria  bacilli.  Kestricted  to  the 
tonsils  alone  their  presence  is  less  certain. 

Occasionally,  in  scarlatinal  angina  or  in  severe  phleg- 
monous  sore-throats,  patches  of  exudate  may  appear  on 
the  uvula  or  borders  of  the  faucial  pillars,  and  still  the 
case  may  not  be  due  to  the  diphtheria  bacilli;  these 
are,  however,  exceptional.  Thick,  grayish  pseudo- 
membranes  which  cover  large  portions  of  the  tonsils, 
soft  palate,  and  nostrils  are  almost  invariably  the 
lesions  produced  by  diphtheria  bacilli. 

The  very  great  majority  of  cases  of  pseudomem- 
branous  or  exudative  laryngitis,  in  the  coast  cities  at 
least,  whether  an  exudate  is  present  in  the  pharynx  or 
not,  are  due  to  the  diphtheria  bacilli.  Cases  in  which 
no  exudate  is  apparent  and  those  in  which  the  laryngeal 
obstruction  is  sudden  and  the  spasmodic  element  is 
marked,  are,  however,  frequently  due  to  the  activity  of 
other  bacteria.  Nearly  all  membranous  affections  of 
the  nose  are  true  diphtheria.  When  the  membrane 
is  limited  to  the  nose  the  symptoms  are,  as  a  rule,  very 
slight;  but  when  the  nasopharynx  is  involved  the 
symptoms  are  usually  grave.  Usually  a  small  area  of 
inflammation  indicates  a  slight  or  moderate  severity, 
and  an  extensive  area  a  severe  infection. 

Most  cases  of  pseudomembranes  and  exudates  entirely 


376  BACTERIOLOGY. 

confined  to  portions  of  the  tonsils  in  adults  are  not  due 
to  the  diphtheria  bacilli,  although  a  few  cases  presenting 
these  symptoms  are.  The  more  complete  the  involve- 
•ment  of  the  tonsils  the  more  apt  the  case  is  to  be  due 
to  them.  Cases  presenting  the  appearances  found  in 
scarlet  fever,  in  which  a  thin,  grayish  membrane  lines 
the  borders  of  the  uvula  and  faucial  pillars,  are  rarely 
diphtheritic.  As  a  rule,  pseudomembranous  inflam- 
mations complicating  scarlet  fever,  syphilis,  and  other 
infectious  diseases  are  due  to  the  activity  of  the  patho- 
genic cocci  and  other  bacteria  induced  by  the  inflamed 
condition  of  the  mucous  membranes  due  to  the  scarla- 
tinal or  other  poison.  But  from  time  to  time  such 
cases,  if  they  have  been  exposed  to  diphtheria,  may  be 
complicated  by  it,  and  in  some  epidemics  mixed  in- 
fection is  common. 

The  Exudate  Due  to  the  Diphtheria  Bacilli  Contrasted 
with  That  Due  to  Other  Bacteria.  As  a  rule,  the  exudate 
in  diphtheria  is  firmly  incorporated  with  the  underlying 
mucous  membrane,  and  cannot  be  removed  without 
leaving  a  bleeding  surface,  at  least  until  convalescence. 
The  tissues  surrounding  the  exudate  are  more  or  less 
inflamed  and  swollen.  Where  other  bacteria  produce 
the  irritant  the  exudate,  except  in  the  cases  due  to  the 
bacillus  described  by  Vincent,  is  usually  loosely  at- 
tached, collected  in  small  masses,  and  easily  removable. 
Exceptions,  however,  occur  in  both  these  diseases,  so 
that  in  true  diphtheria  the  exudate  may  be  easily  re- 
moved, and  in  lesions  due  to  other  bacteria  the  exudate 
may  be  firmly  adherent. 

Paralysis  following  a  pseudomembranous  inflamma- 
tion is  an  almost  positive  indication  that  the  case  was 
one  of  diphtheria,  although  slight  paralysis  has  followed 


DIPHTHERIA  BACILLUS.  377 

in  a  very  few  cases  in  which  careful  cultures  revealed 
no  diphtheria  bacilli.  These,  if  not  true  diphtheria, 
must  be  considered  very  exceptional  cases. 

Bacteriological  Diagnosis.  From  the  above  it  is  appar- 
ent that  fully  developed  characteristic  cases  of  diph- 
theria are  readily  diagnosticated,  but  that  many  of  the 
less  marked,  or  at  an  early  period  undeveloped,  cases 
are  difficult  to  differentiate  the  one  from  the  other. 
In  these  cases  cultures  are  of  the  utmost  value,  since 
they  enable  us  to  isolate  those  in  which  the  bacilli  are 
found,  and  to  give  preventive  injections  of  antitoxin 
to  both  the  sick  and  those  in  contact  with  them,  if  this 
has  not  already  been  done.  As  a  rule,  cultures  do  not 
give  us  as  much  information  as  to  the  gravity  of  the 
case  as  the  clinical  appearances,  for  by  the  end  of 
twenty-four  to  forty-eight  hours  the  extent  of  the  dis- 
ease is  usually  easy  of  determination.  The  reported 
absence  of  bacilli  in  a  culture  must  be  given  weight  in 
proportion  to  the  skill  with  which  the  culture  was  made, 
the  suitableness  of  the  media,  and  the  knowledge  and 
experience  of  the  one  who  examined  it. 

Diphtheria  does  not  occur  without  the  presence  of 
the  diphtheria  bacilli;  but  there  have  been  many  cases 
of  diphtheria  in  which  for  one  or  another  reason  no 
bacilli  were  found  in  the  cultures  by  the  examiner. 
In  many  of  these  cases  later  cultures  revealed  them. 
In  a  convalescent  case  the  absence  of  bacilli  in  any  one 
culture  indicates  that  there  are  certainly  not  many 
bacilli  left  in  the  throat.  Only  repeated  cultures  can 
prove  their  total  absence. 

TECHNIQUE  OF  THE  BACTERIOLOGICAL  DIAGNOSIS. 
Collection  of  the  Blood-serum  and  its  Preparation  for  Use 
in  Cultures.  A  covered  glass  jar  which  has  been  thor- 


378  BACTERIOLOGY. 

oughly  cleansed  with  hot  water  is  taken  to  the  slaughter- 
house and  filled  with  freshly-shed  blood  from  a  calf  or 
sheep.  The  blood  is  received  directly  in  the  jar  as  it 
spurts  from  the  cut  in  the  throat  of  the  animal.  After 
the  edge  of  the  jar  has  been  wiped  it  is  covered  with 
the  lid  and  set  aside,  where  it  may  stand  quietly  until 
the  blood  has  thoroughly  clotted.  The  jar  is  then  car- 
ried to  the  laboratory  and  placed  in  an  ice-chest.  If 
the  jar  containing  the  blood  is  carried  about  before  the 
latter  has  clotted,  very  imperfect  separation  of  the  serum 
will  take  place.  It  is  well  to  inspect  the  blood  in  the 
jar  after  it  has  been  standing  a  few  hours,  and  if  the 
clot  is  found  adhering  to  the  sides,  to  separate  it  by  a 
rod.  The  blood  is  allowed  to  remain  twenty -four  hours 
on  the  ice,  and  then  the  serum  which  surrounds  the  clot 
is  siphoned  off  by  a  rubber  tube  and  mixed  with  one- 
third  its  quantity  of  nutrient  beef  broth,  to  which  1 
per  cent,  glucose  has  been  added.  This  constitutes  the 
Loffler  blood-serum  mixture.  This  is  poured  into  tubes, 
which  should  be  about  four  inches  in  length  and  two- 
thirds  of  an  inch  in  diameter,  having  been  previously 
plugged  witli  cotton  and  sterilized  by  dry  heat  at 
150°  C.  for  one  hour.  Care  should  be  taken  in  filling 
the  tubes  to  avoid  the  formation  of  air-bubbles,  as  they 
leave  a  permanently  uneven  surface  when  the  strum 
has  been  coagulated  by  heat.  To  prevent  this  the  end 
of  the  pipette  or  funnel  which  contains  the  serum  should 
be  inserted  well  into  the  test-tube.  About  2  c.c.  are 
sufficient  for  each  tube.  The  tubes,  having  been  filled 
to  the  required  height,  are  now  to  be  coagulated  and 
sterilized.  They  are  placed  slanted  at  the  proper  angle 
and  then  kept  for  two  hours  at  a  temperature  just  below 
95°  C.  For  this  purpose  a  Koch  serum  coagulator 


DIPHTHERIA  BACILLUS.  379 

(Fig.  22)  or  a  double  boiler  serves  best,  though  a  steam 
sterilizer  will  suffice.  If  the  latter  is  used  a  wire 
frame  must  be  arranged  to  hold  the  tubes  at  the  proper 
inclination,  and  the  degree  of  heat  must  be  carefully 
watched,  as  otherwise  the  temperature  may  go  too 
high,  and  if  the  serum  is  actually  boiled  the  culture 
medium  will  be  spoiled.  After  sterilization  by  this 
process  the  tubes  containing  the  sterile,  solidified  blood- 
serum  can  be  placed  in  covered  tin  boxes  or  stopped 
with  sterile  corks  and  kept  for  months.  The  serum  thus 
prepared  is  quite  opaque  and  firm.  A  mixture  of  blood- 
cells  renders  the  serum  darker,  but  it  is  not  less  useful. 

The  Swab  for  Inoculating  Culture  Tubes.  The  swab 
to  inoculate  the  serum  is  made  as  follows  :  A  stiff,  thin 
iron  rod,  six  inches  in  length,  is  roughened  at  one  end 
by  a  few  blows  of  a  hammer,  and  about  this  end  a  little 
absorbent  cotton  is  firmly  wound.  Each  swab  is  then 
placed  in  a  separate  glass  tube,  and  the  mouths  of  the 
tubes  are  plugged  with  cotton.  The  tubes  and  rods  are 
then  sterilizt  d  by  dry  heat  at  about  150°  C.  for  one 
hour,  and  stored  for  future  use.  Th<se  cotton  swabs 
have  proved  much  more  serviceable  for  making  inocu- 
lations than  platinum  wire  needles,  especially  in  young 
children  and  in  laryngeal  cases.  It  is  easier  to  use  the 
cotton  swab  in  such  cases,  and  it  gathers  up  so  much 
more  material  for  the  inoculation  that  it  has  seemed 
more  reliable. 

For  convenience  and  safety  in  transportation  a  "  cul- 
ture outfit'7  has  been  devised,  which  consists  of  a  small 
wooden  box  containing  a  tube  of  blood-serum,  a  tube 
holding  a  swab,  and  a  record  blank.  These  "  culture 
outfits  "  may  be  carried  or  sent  by  messenger  or  ex- 
press to  any  place  desired. 


380  BACTERIOLOGY. 

Directions  for  Inoculating  Culture  Tubes  with  the  Ex- 
udate.  The  patient  is  placed  in  a  good  light,  and,  if  a 
child,  properly  held.  The  swab  is  removed  from  its 
tube,  and,  while  the  tongue  is  depressed  with  a  spoon, 
is  passed  into  the  pharynx  (if  possible,  without  touch- 
ing the  tongue  or  other  parts  of  the  mouth)  and  is 
rubbed  gently  but  firmly  against  any  visible  membrane 
on  the  tonsils  or  in  the  pharynx,  and  then,  without 
being  laid  down,  the  swab  is  immediately  inserted  in 
the  blood-serum  tube,  and  the  portion  which  has  pre- 
viously been  in  contact  with  the  exudate  is  rubbed  a 
number  of  times  back  and  forth  over  the  whole  sur- 
face of  the  serum.  This  should  be  done  thoroughly, 
but  it  is  to  be  gently  done,  so  as  not  to  break  the  sur- 
face of  the  serum.  The  swab  should  then  be  placed 
in  its  tube,  and  both  tubes,  thin  cotton  plugs  having 
been  inserted,  are  reserved  for  examination  or  sent  to 
the  laboratory  or  collecting  station  (as  in  New  York 
City).  If  sent  to  the  health  department  laboratories 
for  examination  the  blank  forms  of  report  which  usu- 
ally accompany  each  "outfit"  should  be  filled  out  and 
forwarded  with  the  tubes. 

Where  there  is  no  visible  membrane  (it  may  be 
present  in  the  nose  or  larynx)  the  swab  should  be 
thoroughly  rubbed  over  the  mucous  membrane  of  the 
pharynx  and  tonsils,  and  in  the  nasal  cavities,  and  a 
culture  made  from  these.  In  very  young  children  care 
should  be  taken  not  to  use  the  swab  when  the  throat 
contains  food  or  vomited  matter,  as  then  the  bacterio- 
logical examination  is  rendered  more  difficult.  Under 
no  conditions  should  any  attempt  be  made  to  collect 
the  material  shortly  after  the  application  of  strong 


DIPHTHERIA  BACILLUS.  381 

disinfectants  (especially  solutions  of  corrosive  subli- 
mate) to  the  throat. 

Examination  of  Cultures.  The  culture  tubes  which 
have  been  inoculated,  as  described  above,  are  kept  in 
an  incubator  at  37°  C.  for  twelve  hours,  and  are  then 
ready  for  examination.  When  great  haste  is  required, 
even  five  hours  will  often  suffice  for  a  sufficient  growth 
of  bacteria  for  a  skilled  examiner  to  decide  as  to  the 
presence  or  absence  of  the  bacilli.  On  inspection  it 
will  be  seen  that  the  surface  of  the  blood-serum  is 
dotted  with  numerous  colonies,  which  are  just  visible. 
No  diagnosis  can  be  made  from  simple  inspection ;  if, 
however,  the  serum  is  found  to  be  liquefied  or  shows 
other  evidences  of  contamination  the  examination  will 
probably  be  unsatisfactory. 

In  order  to  make  a  microscopical  preparation  a  clean 
platinum  needle  is  inserted  in  the  tube  and  quite  a 
large  number  of  colonies  are  swept  with  it  from  the 
surface  of  the  culture  medium,  a  part  being  selected 
where  small  colonies  only  are  found.  A  sufficient 
amount  of  the  bacteria  adherent  to  the  needle  are 
washed  off  in  the  drop  of  water  previously  placed  on 
the  cover-glass  and  smeared  over  its  surface.  The 
bacteria  on  the  glass  are  then  allowed  to  dry  in  the 
air.  The  cover-glass  is  then  passed  quickly  through 
the  flame  of  a  Bunsen  burner  or  alcohol  lamp,  three 
times  in  the  usual  way,  covered  with  a  few  drops  of 
Loffler's  solution  of  alkaline  methylene-blue,  and  left 
without  heating  for  ten  minutes.  It  is  then  rinsed  off 
•  in  clear  water,  dried,  and  mounted  in  balsam.  When 
other  methods  of  staining  are  desired  they  are  carried 
out  in  the  proper  way. 

In  the  great  majority  of  cases  one  of  two  pictures 


382  BACTERIOLOGY. 

will  be  seen  with  the  1/12  oil  immersion  lens — either 
an  enormous  number  of  characteristic  Loffler-ba(  illi, 
with  a  moderate  number  of  cocci,  or  a  pure  culture  of 
cocci,  mostly  in  pairs  or  short  chains  (see  Streptococ- 
cus). In  a  few  cases  there  will  be  an  approximately 
even  mixture  of  Loffler  bacilli  and  cocci,  and  in  others 
a  great  excess  of  cocci.  Beside  these,  there  will  be 
occasionally  met  preparations  in  which,  with  the  cocci, 
there  are  mingled  bacilli  more  or  less  resembling  the 
Loffler  bacilli.  These  bacilli,  which  are  usually  of 
the  pseudodiphtheria  type  of  bacilli  (see  Fig.  46),  are 
especially  frequent  in  cultures  from  the  nose. 

In  not  more  than  one  case  in  twenty  will  there  be 
any  serious  difficulty  in  making  the  diagnosis,  if  the 
serum  in  the  tube  was  moist  and  had  been  properly 
inoculated.  In  such  a  case  another  culture  must  be 
made  or  the  bacilli  plated  out  and  tested  in  pure 
culture. 

Direct  Microscopical  Examination  of  the  Exudate. 
An  immediate  diagnosis  without  the  use  of  cultures 
is  often  possible  from  a  microscopical  examination  of 
the  exudate.  This  is  made  by  smearing  a  slide  or 
cover-^lass  with  a  little  of  the  exudate  from  the  swab, 
drying,  heating,  staining,  and  examining  it  microscop- 
ically. This  examination,  however,  is  much  more  diffi- 
cult, and  the  results  are  more  uncertain  than  when  the 
covers  are  prepared  from  cultures.  The  bacilli  from 
the  membrane  are  usually  less  typical  in  appearance 
than  those  found  in  cultures,  and  they  are  mixed  with 
fibrin,  pus,  and  epithelial  cells.  They  may  also  be 
very  few  in  number  in  the  parts  reached  by  the  swab, 
or  bacilli  may  be  met  with  which  closely  resemble  the 
Loffier  bacilli  in  appearance,  but  which  differ  greatly 


DIPHTHERIA  BACILLUS.  383 

in  growth  and  in  other  characteristics,  and  have  abso- 
lutely no  connection  with  them.  When  in  a  smear 
containing  mostly  cocci  a  few  of  these  doubtful  bacilli 
are  present,  it  is  impossible  either  to  exclude  or  to  make 
the  diagnosis  of  diphtheria  with  certainty.  Although 
in  some  cases  this  immediate  examination  may  be  of 
the  greatest  value,  it  is  not  a  method  suitable  for  gen- 
eral use,  and  should  always  be  controlled  by  cultures. 

Animal  Inoculation  as  a  Test  of  Virulence.  If  the 
determination  of  the  virulence  of  the  bacilli  found  is 
of  importance,  animal  inoculations  must  be  made.  Ex- 
periments on  animals  form  the  only  method  of  deter- 
mining with  certainty  the  virulence  of  the  diphtheria 
bacillus.  For  this  purpose,  alkaline  broth  cultures  of 
forty-eight  hours7  growth  should  be  used  for  the  sub- 
cutaneous inoculation  of  guinea-pigs.  The  amount 
injected  should  not  be  more  than  one-fifth  per  cent,  of 
the  body-weight  of  the  animal  inoculated  unless  con- 
trols with  antitoxin  are  made.  In  the  large  majority 
of  cases,  when  the  bacilli  are  virulent,  this  amount 
causes  death  within  seventy-two  hours.  At  the  autopsy 
the  characteristic  lesions  already  described  are  found. 
Bacilli  which  in  cultures  and  in  animal  experiments 
have  shown  themselves  to  be  characteristic  may  be 
regarded  for  practical  purposes  as  certainly  true  diph- 
theria bacilli,  and  as  capable  of  producing  diphtheria 
in  man  under  favorable  conditions. 

For  an  absolute  test  of  specific  virulence  antitoxin 
must  be  used.  A  guinea-pig  is  injected  with  antitoxin, 
and  then  this  and  a  control  animal,  with  double  the 
fatal  dose  of  a  broth  culture  of  the  bacilli  to  be  tested; 
if  the  guinea-pig  which  received  the  antitoxin  lives, 
while  the  control  dies,  it  was  surely  a  diphtheria  bacil- 


384  BACTERIOLOGY. 

lus  which  killed  by  means  of  diphtheria  toxin — or,  in 
other  words,  not  simply  a  virulent  bacillus,  but  a  viru- 
lent diphtheria  bacillus.  When  the  bacilli  to  be  tested 
grow  poorly  in  the  simple  nutrient  bouillon  they  should 
be  grown  in  bouillon  to  which  one-third  its  quantity  of 
ascitic  fluid  has  been  added.  Quite  a  number  of  bacilli 
have  been  met  with  which  killed  250  gramme  guinea- 
pigs  in  doses  of  2  to  15  c.c.,  and  yet  were  unaffected 
by  antitoxin.  These  bacilli,  though  slightly  virulent 
to  guinea-pigs,  produce  no  diphtheria  toxin,  and  so 
cannot,  to  the  best  of  our  belief,  produce  diphtheria  in 
man. 


CHAPTER  XXII. 

THE   BACILLUS   OF   TETANUS. 

IN  1884,  Nicolaier,  a  student  in  Fliigge's  Institute, 
produced  tetanus  in  mice  and  rabbits  by  the  subcutaneous 
inoculation  of  particles  of  garden  earth,  and  showed  that 
the  disease  was  transmissible  by  inoculation  from  these 
animals  to  others.  Carle  and  Rattoiie,  in  1884,  demon- 
strated the  infectious  nature  of  tetanus  as  it  occurs  in 
man.  Finally,  Kitasato,  in  1889,  obtained  the  bacillus 
of  tetanus  in  pure  culture  and  described  his  method  of 
obtaining  it  and  its  biological  characters. 

The  tetanus  bacillus  occurs  in  nature  as  a  common 
inhabitant  of  the  soil,  at  least  in  places  where  manure 
has  been  thrown,  being  abundant  in  many  localities, 
not  only  in  the  superficial  layers,  but  also  at  the  depth 
of  several  feet.  It  has  been  found  in  many  different 
substances  and  places — in  hay-dust,  in  horse  and  cow 
manure,  in  the  mortar  of  old  masonry,  in  the  dust  from 
horses'  hair,  in  the  dust  in  rooms  of  houses,  barracks, 
and  hospitals,  in  the  air,  and  in  the  arrow  poison  of 
certain  savages  in  the  New  Hebrides,  who  obtained  it 
by  smearing  the  arrow-heads  with  dirt  from  crab  holes 
in  the  swamps. 

Morphology.  Motile,  slender  rods,  with  rounded  ends, 
0.3//  to  0.5/jt.  in  diameter  by  2/j.  to  4fj.  in  length,  usually 
occurring  singly,  but,  especially  in  old  cultures-,  often 
growing  in  long  threads.  They  form  round  spores, 

25 


386  BACTERIOLOGY. 

thicker  than  the  cell  (from  I  ft  to  1.5//  in  diameter), 
occupying  one  of  its  extremities  and  giving  to  the  rods 
the  appearance  of  small  pins  (Fig.  48).  It  is  stained 
with  the  ordinary  aniline  dyes,  and  is  not  decolorized 
by  Gram's  method.  The  spores  may  be  demonstrated 
by  double-staining  with  ZiehPs  method. 

FIG.  48. 


Tetanus  bacilli  with  spores  in  distended  ends.    X  1100  diameters. 

Biology.  An  anaerobic,  liquefying,  motile  (though  not 
very  actively  motile)  bacillus.  Forms  spores,  and  in 
the  spore  stage  it  is  not  motile.  It  does  not  grow  at 
temperatures  below  14°  C.,  but  grows  slowly  at  tem- 
peratures from  20°  to  24°  C.,  and  best  at  37°  C.,  when 
it  rapidly  forms  spores.  It  will  not  grow  in  the  pres- 
ence of  oxygen  or  carbon  dioxide  gas,  but  grows  well  in 
an  atmosphere  of  pure  hydrogen. 

The  bacillus  of  tetanus  grows  in  ordinary  nutrient 
gelatin  and  agar  of  a  slightly  alkaline  reaction.  The 
addition  to  the  media  of  1.5  per  cent,  of  glucose  causes 
the  development  to  be  more  rapid  and  abundant.  It 
also  grows  abundantly  in  alkaline  bouillon  under  an 
atmosphere  of  hydrogen. 


THE  BACILLUS  OF  TETANUS.  387 

Its  growth  in  the  animal  organism  is  comparatively 
scanty,  and  is  usually  associated  with  other  bacteria; 
hence,  it  is  difficult  to  obtain  it  in  pure  culture.  The 
method  of  procedure  proposed  by  Kitasato,  which,  how- 
ever, is  not  always  successful,  consists  in  inoculating  an 
agar  tube  with  the  tetanus-bearing  material  (pus  from 
the  inoculation  wound),  keeping  this  for  twenty-four  to 
forty-eight  hours  at  a  temperature  of  37°  C.,  and,  after 
the  tetanus  spores  have  formed,  heating  it  for  about  an 
hour  at  80°  C.,  to  destroy  the  associated  bacteria.  The 
spores  of  the  tetanus  bacillus  being  able  to  survive  this 
exposure,  anaerobic  cultures  are  then  made  in  the  usual 
way,  and  the  tetanus  colonies  thus  isolated.  The  fur- 
ther development  is  unattended  with  difficulty.  On 
gelatin  plates  the  colonies  develop  slowly;  they  resemble 
somewhat  the  colonies  of  the  bacillus  subtilis,  and  have 
a  dense,  opaque  centre  surrounded  by  fine,  diverging 
rays.  Liquefaction  takes  place  more  slowly,  however, 
than  with  the  bacillus  subtilis,  and  the  resemblance  to 
these  colonies  is  soon  lost.  In  old  cultures  the  entire 
mass  is  made  iip  of  a  number  of  fine  threads,  and  the 
colonies  are  not  unlike  those  of  the  common  mould. 

The  colonies  on  agar  are  quite  characteristic  (San- 
felice).  To  the  naked  eye  they  present  the  appearance 
of  light,  fleecy  clouds;  under  the  microscope,  a  tangle 
of  fine  threads.  The  extreme  fineness  of  the  threads 
enables  them  to  be  distinguished  from  the  colonies  of 
-other  anaerobes. 

The  stab  cultures  in  gelatin  exhibit  the  appearance  of 
a  cloudy,  linear  mass,  with  prolongations  radiating  into 
the  gelatin  from  all  sides.  Liquefaction  takes  place 
slowly,  generally  with  the  production  of  gas.  In  stab 
cultures  in  agar  a  growth  occurs  not  unlike  in  structure 


388  BACTERIOLOGY. 

that  of  a  miniature  pine-tree.  Alkaline  bouillon  is  ren- 
dered somewhat  turbid  by  the  growth  of  the  tetanus 
bacillus.  In  all  cases  a  production  of  gas  results,  accom- 
panied by  a  characteristic  and  very  disagreeable  empy- 
reumatic  odor.  It  also  grows  in  acid  culture  media, 
but  of  itself  produces  no  acid.  It  develops  in  milk 
without  coagulating  it,  and  starch  is  not  hydrated  by 
it  in  its  growth  (Sanfelice). 

The  spores  of  the  tetanus  bacillus  are  very  resistant 
to  outside  influences;  they  retain  their  vitality  for 
months  and  years  in  a  desiccated  condition,  and  are 
not  destroyed  in  two  and  a  half  months  when  present  in 
putrefying  material  (Turco).  They  withstand  an  ex- 
posure of  one  hour  to  80°  C.,  but  are  killed  by  an  ex- 
posure of  five  minutes  to  100°  C.  in  the  steam  sterilizer. 
They  resist  the  action  of  5  per  cent,  carbolic  acid  for 
ten  hours,  but  succumb  when  exposed  to  it  for  fifteen 
hours.  A  5  per  cent,  solution  of  carbolic  acid,  how- 
ever, to  which  0.5  per  cent,  of  hydrochloric  acid  has 
been  added,  destroys  them  in  two  hours.  When  acted 
upon  for  three  hours  by  bichloride  of  mercury  (1  :  1000) 
they  are  killed,  and  in  thirty  minutes  when  0.5  per  cent. 
HC1  is  added  to  the  solution.  If  the  solution  contains 
1  :  1000  bichloride,  with  5  per  cent,  carbolic  and  a  0.5 
per  cent.  HC1,  the  spores  are  killed  in  ten  minutes. 
Silver  nitrate  solutions  destroy  the  spores  in  one  minute 
in  1  per  cent,  solution  and  in  five  minutes  in  1  :  1000 
solution. 

Pathogenesis.  In  mice,  guinea-pigs,  rabbits,  rats, 
horses,  goats,  and  a  number  of  other  animals  inocula- 
tions of  pure  cultures  of  the  tetanus  bacillus  cause  typi- 
cal .tetanus  after  an  incubation  of  from  one  to  three 
days.  A  mere  trace — only  as  much  as  remains  cling- 


THE  BACILLUS  OF  TETANUS.  389 

ing  to  a  platinum  needle — of  an  old  culture  is  often 
sufficient  to  kill  very  susceptible  animals  like  mice  and 
guinea-pigs.  Other  animals  require  a  larger  amount. 
Birds  are  but  little  susceptible,  and  fowls  scarcely  at 
all.  It  is  a  remarkable  fact  that  an  amount  of  toxin 
sufficient  to  kill  a  hen  would  suffice  to  kill  500  horses. 
On  the  inoculation  of  less  than  a  fatal  dose  in  test- 
animals  a  local  tetanus  may  be  produced,  which  lasts 
for  days  and  weeks  and  then  ends  in  recovery.  On 
killing  the  animal  there  is  found  at  autopsy,  just  at  the 
point  of  inoculation,  a  hemorrhagic  spot,  and  no  changes 
here  or  in  the  interior  organs  other  than  these.  A  few 
tetanus  bacilli  may  be  detected  locally  with  great  diffi- 
culty, often  none  at  all;  possibly  a  few  may  be  found 
in  the  region  of  the  lymphatic  glands.  From  this 
scanty  occurrence  of  bacilli  the  conclusion  has  been 
reached  that  the  bacilli  of  tetanus,  when  inoculated  in 
pure  culture,  do  not  multiply  in  the  living  body,  but 
only  produce  lesions  through  the  absorption  of  the 
poison  which  they  produce  at  the  point  of  infec- 
tion. These  authors  also  found  that  pure  cultures  of 
tetanus,  after  the  germs  had  sporulated  and  the  toxins 
had  been  destroyed  by  heat,  could  be  injected  into 
animals  without  producing  tetanus.  Even  one  or  two 
millions  of  spores,  if  deprived  of  the  toxins,  proved 
harmless  to  guinea-pigs,  and  from  15  to  30  c.c.  of 
broth  cultures  were  harmless  to  rabbits.  But  if  a  cul- 
ture of  non-pathogenic  organisms  was  injected  simul- 
taneously with  the  spores,  or  if  there  was  an  effu- 
sion of  blood  at  the  point  of  injection,  or  if  there  was 
a  previous  bruising  of  the  tissues,  the  animals  surely 
died  of  tetanus.  Even  irritating  foreign  bodies  were 
introduced  along  with  the  spores  deprived  of  their 


390  BACTERIOLOGY. 

toxins,  and  tetanus  did  not  develop;  bat  if  the  wounds 
containing  the  foreign  bodies  became  infected  with 
other  bacteria,  tetanus  developed  and  the  animals  died. 
From  these  experiments  it  seems  that  a  mixed  infection 
is  necessary  to  the  development  of  tetanus  when  the 
infection  is  produced  by  spores. 

This  fact  i's  of  the  greatest  importance  in  natural 
tetanus.  Here  the  infection  may  be  considered  as 
prubably  invariably  produced  by  the  bacilli  in  their 
spore  state,  and  the  conditions  favoring  infection  are 
almost  always  present.  A  wound  of  some  kind  has 
occurred,  penetrating  at  least  through  the  skin,  though 
perhaps  of  a  most  trivial  character,  such  as  might  be 
caused  by  a  dirty  splinter  of  wood,  and  the  bacilli  or 
their  spores  are  thus  introduced  from  the  soil  in  which 
they  are  so  widely  distributed.  If  in  any  given  case, 
the  tissues  being  healthy,  the  ordinary  saprophytic 
germs  are  killed  by  proper  disinfection  at  once,  a 
mixed  infection  does  not  take  place,  and  tetanus  will 
not  develop.  If,  however,  the  tissues  infected  be 
badly  bruised  or  lacerated,  the  spores  may  develop 
and  produce  the  disease.  With  regard  to  the  persist- 
ence of  tetanus  spores  upon  objects  where  they  have 
found  a  resting-place,  Henrijean  reports  that  by  means 
of  a  splinter  of  wood  which  had  once  caused  tetanus 
he  was  able  after  eleven  years  to  again  cause  the  dis- 
ease by  inoculating  an  animal  with  the  same  splinter. 
The  bacilli  of  tetanus  are  apparently  more  numerous 
in  certain  localities  than  in  others — for  example,  some 
parts  of  Long  Island  and  New  Jersey,  which  have 
become  notorious  for  the  number  of  cases  of  tetanus 
caused  by  small  wounds — but  they  are  very  generally 
distributed,  as  the  experiments  on  animals  inoculated 


THE  BACILLUS  OF  TETANUS.  391 

with  garden  earth  have  shown,  and  are  fairly  common 
in  New  York  City. 

Man  and  almost  all  domestic  animals  are  subject  to 
tetanus.  On  examination  of  an  infected  individual 
very  little  local  evidence  of  the  disease  can  be  discov- 
ered. Generally  at  the  point  of  infection,  if  there  is 
an  external  wound,  some  pus  is  to  be  seen,  in  which, 
along  with  numerous  other  bacteria,  tetanus  bacilli  or 
their  spores  may  be  found.  By  successive  inoculation 
of  this  pus  in  susceptible  animals  the  disease  can  often  be 
reproduced  for  from  four  to  five  generations;  but  some- 
times there  is  a  break  in  the  chain,  which  proves  that 
in  such  cases  the  infection  has  been  brought  about  less 
by  the  bacilli  than  by  the  toxin  which  was  transmitted 
with  them. 

Not  only  traumatic  tetanus,  but  also  all  the  various 
forms  of  tetanus,  are  now  conceded  to  be  produced  by 
the  tetanus  bacillus — puerperal  tetanus,  tetanus  neona- 
torum,  and  idiopathic  and  rheumatic  tetanus.  In 
tetanus  neonatorum  and  puerperal  tetanus  the  infection 
is  introduced  through  the  navel  and  the  inner  surface 
of  the  uterus.  It  should  be  borne  in  mind,  however, 
that  when  there  is  no  external  and  visible  wound  there 
may  be  an  internal  one.  Carbone  and  Perrero  report 
a  case  of  so-called  rheumatic  tetanus  in  which  attenu- 
ated forms  of  tetanus  bacilli  were  found  in  the  bronchial 
secretions.  These  bacilli  possessed  the  morphological 
and  cultural  peculiarities  of  the  tetanus  bacilli,  but 
they  did  not  produce  toxin.  Similar  anaerobes  have 
been  found  in  meat-juices  and  in  the  soil.  The  bacilli 
found  in  the  bronchial  secretions,  therefore,  may  have 
been  tetanus  bacilli  which,  owing  to  certain  conditions, 
had  lost  their  virulence,  just  as  we  know  it  to  happen 


392  BACTERIOLOGY. 

in  diphtheria.  It  may  well  be  supposed  that  the  mucous 
membranes  of  the  bronchi,  and  other  similar  mem- 
branes, in  a  condition  of  catarrhal  inflammation,  may 
be  more  susceptible  to  tetanus  infection  than  they 
normally  are. 

Tetanus  Toxin.  It  is  evident  from  the  localization  of 
the  tetanus  bacilli  at  the  point  of  inoculation  and  their 
slight  multiplication  at  this  point  that  they  owe  their 
action  to  the  production  of  a  powerful  toxin.  While 
there  are  a  few  cases  on  record  in  which  the  bacilli 
have  been  found  in  the  tissues  of  the  animal  body 
other  than  the  point  of  infection,  the  fact  remains  that 
in  the  vast  majority  of  cases  the  tetanus  bacillus  is 
localized.  This  toxin  can  be  readily  separated  from 
cultures  by  filtration.  One-hundredth  of  a  milligramme 
of  an  eight-day  filtered  bouillon  culture  is  sufficient,  as 
a  rule,  to  kill  a  mouse.  From  this  filtrate,  however, 
the  active  toxic  substance  has  been  obtained  in  a  much 
more  concentrated  form.  The  purified  and  dried  tetanus 
toxin  prepared  by  Brieger  and  Cohn  was  surely  fatal  to 
a  15  gramme  mouse  in  a  dose  of  0.00000005  gramme. 
Reckoning  according  to  the  body-weight  of  75  kilo- 
grammes, or  175  pounds,  it  would  require  but  0.00023 
gramme,  or  0.23  milligramme  of  this  toxin,  to  prove 
fatal  to  a  man.  By  comparing  this  with  snake- 
poison,  Calmette  has  found  that  dried  cobra  venom 
requires  0.25  milligramme  to  kill  a  rabbit  of  4  kilo- 
grammes' weight,  and  according  to  body-weight,  it 
would  require  4.375  milligrammes  to  kill  a  man  of  70 
kilogrammes.  As  the  fatal  dose  of  atropine  for  an 
adult  is  130  milligrammes,  of  strychnine  from  30  to 
100  milligrammes,  and  of  anhydrous  prussic  acid  54 
milligrammes,  the  appalling  strength  of  the  tetanus  toxin 


THE  BACILLUS  OF  TETANUS.  393 

can  readily  be  appreciated  (Lambert).  What  the  true 
composition  and  constitution  of  the  tetanus  poisons  are 
is  unknown.  It  has  been  shown,  however,  that  it  pos- 
sesses neither  the  characteristics  of  an  alkaloid  (ptomain) 
nor  of  an  albuminous  body  (toxalbumin);  it  is  largely 
precipitated  from  fluids  saturated  with  ammonium  sul- 
phate. 

The  quantity  of  the  toxin  produced  varies,  even 
when  derived  from  one  and  the  same  culture,  according 
to  the  age  of  the  culture,  its  composition,  reaction,  etc. ; 
and  partly  it  is  due  to  the  extreme  sensitiveness  of  the 
toxin,  which  cannot  bear  keeping  any  length  of  time  or 
exposure  to  light,  being  sensibly  affected  by  most  chem- 
ical reagents  and  destroyed  by  heating  to  55°  to  60°  C. 
for  any  length  of  time.  It  retains  its  strength  best  in 
the  dry  state. 

Some  authors  (Kitasato  and  Sanfelice)  have  main- 
tained that  the  tetanus  cultures  retain  their  viru- 
lence unaltered;  others,  again,  have  observed  consider- 
able alteration  in  toxicity.  Righi,  for  instance,  has 
observed  that  the  tetanus  bacillus  cultivated  under 
aerobic  conditions  may  entirely  lose  its  virulence. 
Certain  chemical  agents  also  produce  on  cultures  of  the 
tetanus  bacillus  an  attenuation  of  virulence,  if  only  a 
temporary  one. 

The  Action  of  Tetanus  Toxin  in  the  Body.  The  parts 
first  to  be  affected  with  tetanus  are  in  about  one- third  of 
the  cases  in  man,  and  usually  in  animals  the  muscles 
lying  in  the  vicinity  of  the  inoculation — for  instance, 
the  hind  foot  of  a  mouse  inoculated  on  that  leg  is  first 
affected,  then  the  tail,  the  other  foot,  the  back  and 
chest  muscles  on  both  sides,  and  the  forelegs,  until 
finally  there  is  a  general  tetanus  of  the  entire  body. 


394  BACTERIOLOGY. 

In  mild  cases,  or  when  a  dose  too  small  to  be  fatal 
has  been  received,  the  tetanic  spasm  may  remain  con- 
fined to  the  muscles  adjacent  to  the  point  of  inocula- 
tion or  infection.  According  to  Gumprecht,  the  action 
of  tetanus  depends  upon  an  increased  reflex  excita- 
bility, as  in  strychnine-poisoning;  but  it  is  different 
from  strychnine  in  its  mode  of  distribution,  and  prob- 
ably takes  place  chiefly  through  the  nervous  system,  as 
in  rabies.  This  view  is  supported  by  Brunner,  Brusch- 
ettini,  and  others.  Beck  has  described  a  peculiar  degen- 
eration in  the  motor  cells  of  the  cord  in  animals  killed 
by  tetanus.  This  degeneration  does  not  seem  to  attack 
the  entire  cells,  but  only  a  peripheral  part,  and  seems 
to  be  confined  chiefly  to  the  body  of  the  cell,  usually 
leaving  the  nucleus  intact.  Only  very  late  do  the 
nucleus  and  the  nucleolus  take  part  in  the  changes. 
The  changes  consist  in  a  swelling  of  the  cell  and  a 
homogeneous  or  finely  granular  degeneration  with  a 
swelling,  and,  finally,  coarse  lumping  together  of  the 
chromatin  This  is  especially  evident  at  the  tiny  emi- 
nence from  which  the  axis-cylinder  arises  and  in  the 
axis-cylinder  itself.  Beck  considers  this  as  proving 
that  the  poison  travels  along  the  axis-cylinder,  and 
that,  as  the  nucleus  is  the  last  portion  affected,  the 
change  is  not  a  necrosis  but  only  a  modification  of 
cell  function. 

But  there  is  also,  in  addition,  undoubtedly  a  diffusion 
of  the  poison  by  means  of  the  blood  and  lymph.  The 
blood  usually  contains  the  poison,  as  has  been  proved 
experimentally  on  animals.  Neisser  showed  that  the 
blood  of  a  tetanic  patient  was  capable  of  inducing 
tetanus  in  animals  when  injected  subcutaneously. 
Kitasato  also  found  the  serous  exudates  of  the  pleural 


THE  BACILLUS  OF  TETANUS.  395 

and  pericardial  cavities  as  well  as  the  blood  of  tetanic 
animals  would  cause  tetanus  when  transferred  to  other 
animals.  Kahlmeyer,  Bruschettini,  and  others  have 
obtained  similar  results.  The  toxin  has  also  been 
demonstrated  in  the  urine  when  large  amounts  have 
been  inoculated. 

Courmont  and  Doyon  believe  that  the  so-called  toxin 
elaborated  by  the  tetanus  bacillus  is  not  the  true  poison, 
but  is  a  ferment  which  forms  from  the  poison  in  the 
body  at  the  expense  of  the  organism,  and  is  found 
in  the  blood,  sometimes  in  the  urine,  and  in,  especial 
abundance  in  tetanized  muscles.  The  action  of  tetanus 
toxin  is  never  suddenly  produced,  though  when  once 
formed  its  absorption  is  rapid,  but  always  requires  a 
certain  period  of  incubation.  These  authors  hold  that 
the  substances  produced  by  the  tetanus  bacillus  must 
undergo  a  chemical  change  in  the  body,  because  after 
it  is  formed  in  the  tissues  it  can  be  extracted  from 
them  by  boiling,  and  when  injected  into  other  animals 
causes  immediate  tetanic  symptoms  without  any  period 
of  incubation.  But  other  observers  repeating  these 
experiments  have  failed  to  confirm  Courmont  and 
Doyon' s  results,  and  appear  to  have  proved  their 
theory  to  be  untenable. 

Tetanus  Antitoxin.  Behring  and  Kitasato  were  the 
first  to  show  the  possibility  of  immunizing  animals 
against  tetanus  infection.  Here  the  question  of  immu- 
nity against  infection  does  not  consist  in  producing  an 
increased  power  of  resistance  against  the  development 
of  the  infecting  agent,  as  is  the  case  in  most  infectious 
diseases,  but  similar  to  diphtheria,  in  bringing  about 
an  immunity  to  the  effects  of  the  tetanus  toxin.  The 
bacillus  of  tetanus,  as  we  have  seen,  does  not  belong 


396  BACTERIOLOGY. 

to  the  septicsemic  class  of  organisms  which  spreads 
through  the  body,  and  by  their  growth  and  increase 
produce  their  effects,  but,  on  the  contrary,  remains 
localized  at  the  original  point  of  infection.  It  pro- 
duces, however,  in  its  growth  a  most  powerful  toxin. 
The  treatment  of  tetanus  is,  therefore,  directed  against 
the  production  of  toxin  and  its  neutralization  in  the 
body.  The  methods  originally  proposed  by  Behring  and 
by  Roux  for  producing  a  curative  serum  consisted 
chiefly  in  weakening  the  tetanus  toxin  by  means  of 
chemical, disinfectants  (iodine  trichloride,  Gram's  and 
LugoPs  solutions),  so  that  when  inoculated  into  the 
test-animals  they  produced  comparatively  little  reac- 
tion. At  the  present  time  we  inject  the  pure  unaltered 
toxin  either  alone  in  small  doses  or  along  with  anti- 
toxin. After  the  first  dose  of  toxin  the  animals  acquire 
a  certain  tolerance  which  enables  them  to  stand  a  dose 
of  a  less  attenuated  toxin  or  of  a  greater  amount  of  un- 
changed toxin.  Thus  by  gradually  increasing  the  doses 
or  the  strength  of  the  toxin  administered,  the  animals 
are  finally  able  to  bear  injections  of  large  quantities  of 
the  strongest  toxin. 

These  immunizing  experiments  in  tetanus  have  borne 
practical  fruit,  for  it  was  through  them  that  the  prin- 
ciple of  serum-therapeutics  first  became  known — the 
protective  and  curative  effects  of  the  blood -serum  of 
immunized  animals.  It  was  thus  shown  that  animals 
could  be  protected  from  tetanus  infection  by  the  pre- 
vious or  simultaneous  injection  of  tetanus  antitoxin, 
provided  that  such  antitoxic  serum  was  obtained  from 
a  thoroughly  immunized  animal;  and  from  this  it  was 
assumed  that  the  same  result  could  be  produced  in  natu- 
ral tetanus  in  man;  but,  unfortunately,  the  conditions 


THE  BACILLUS  OF  TETANUS.  397 

in  the  natural  disease  are  very  much  less  favorable,  in- 
asmuch as  treatment  is  usually  commenced  not  shortly 
after  the  infection  has  taken  place,  but  often  only  on 
the  appearance  of  tetanic  symptoms,  when  the  poison 
has  already  diffused  itself  through  the  body. 

Tetanus  Antitoxin.  The  tetanus  antitoxin  is  developed 
in  the  same  manner  as  the  diphtheria  antitoxin — by 
inoculating  the  tetanus  toxin  in  increasing  doses  into 
horses.  The  toxin  is  produced  in  bouillon  cultures 
grown  anaerobically.  After  ten  or  fifteen  days  the 
culture  fluid  is  filtered  through  porcelain,  and  the  germ- 
free  filtrate  is  used  for  the  inoculations.  The  horses 
receive  half  a  c.c.  as  the  initial  dose  of  a  toxin  of  which 
1  c.c.  kills  250,000  grammes  of  guinea-pig,  and  along 
with  this  a  sufficient  amount  of  antitoxin  to  neutralize 
it.  In  five  days  this  dose  is  doubled,  and  then  every 
five  to  seven  days  larger  amounts  are  given.  The  dose 
is  increased,  as  rapidly  as  the  horse  s  can  stand  it,  until 
they  support  700  to  800  c.c.  or  more  at  a  single  injec- 
tion. After  some  months  of  this  treatment  the  blood 
of  the  horse  contains  the  antitoxin  in  sufficient  amount 
for  therapeutic  use.  When  the  animals7  temperatures 
are  normal  and  they  have  recovered  from  the  dose  of 
toxin  last  given,  they  are  bled  into  sterile  flasks  and 
the  serum  collected. 

Technique  of  Testing  Antitoxin  Serum  for  Value  in 
Antitoxin.  Tetanus  antitoxin  is  tested  exactly  in  the 
same  manner  as  diphtheria  antitoxin,  except  that  the 
standard  unit  is  different.  The  test  toxin  used  in  the 
German  method  is  one  of  which  1  gramme  destroys 
150,000,000  grammes  of  mouse.  This  is  dissolved  in 
33  J  c.c.  of  10  per  cent.  NaCl  solution.  Ten  times  the 
amount  of  antitoxic  serum  which  neutralizes  1  c.c.  of 


398  BACTERIOLOGY. 

this  dilution  of  the  test  toxin  contains  one  unit  of  anti- 
toxin. In  the  French  method  the  amount  of  antitoxin 
which  is  required  to  protect  a  mouse  from  a  dose  of 
toxin  sufficient  to  kill  in  four  days  is  determined,  and 
the  strength  of  the  antitoxin  is  stated  by  determining 
the  amount  of  serum  required  to  protect  one  gramme 
of  animal.  If  0.001  c.c.  protected  a  10  gramme  mouse 
the  strength  of  that  serum  would  be  1 : 10,000.  Guinea- 
pigs  are  sometimes  used  in  place  of  mice.  Knorr's  toxin 
is  preserved  by  precipitating  it  with  saturated  ammo- 
nium sulphate  and  drying  and  preserving  the  precipi- 
tate in  sealed  tubes.  As  required,  it  is  dissolved  in  10 
per  cent,  salt  solution,  as  above  stated.  For  small 
testing  stations  the  best  way  is  to  obtain  some  freshly 
standardized  antitoxin  and  compare  serums  with  this. 

The  Persistence  of  Antitoxin  in  the  Blood.  Ransom 
has  recently  shown  that  the  tetanus  antitoxin  is  elimi- 
nated just  about  as  rapidly  from  the  blood  of  an  animal 
when  produced  by  toxin  injections  as  when  injected 
with  antitoxin,  so  long  as  the  serum  was  from  an  ani- 
mal of  the  same  race.  When  from  a  different  race,  it 
is  much  more  quickly  eliminated.  From  this  we  see 
a  possible  explanation  of  the  fact  that  immunity  in 
man,  due  to  an  injection  of  the  antitoxic  serum  of  the 
horse,  is  less  persistent  than  immunity  conferred  by  an 
attack  of  the  disease. 

He  found  some  interesting  facts  in  testing  the  anti- 
toxic values  of  the  serum  of  an  immunized  mare,  of  its 
foal,  and  of  the  milk.  The  foal's  serum  was  one-third 
the  strength  of  the  mare's,  and  one  hundred  and  fifty 
times  that  of  the  mare's  milk.  In  two  months  the 
mare's  serum  lost  two-thirds  in  antitoxic  strength,  the 
foal's  five-sixths,  and  the  milk  one-half.  Injections  of 


THE  BACILLUS  OF  TETANUS.  399 

toxin  were  then  given  the  mare,  so  that  it  doubled  its 
original  strength  in  one  month.  The  milk  increased 
eightfold,  but  the  foal's  continued  to  lose  in  antitoxin, 
although  it  was  feeding  on  the  antitoxic  milk. 

Eesults  of  the  Antitoxin  Treatment  in  Tetanus.  Tetanus 
is  a  comparatively  rare  disease  both  in  man  and  animals, 
though  in  some  localities  it  is  more  common  than  in 
others.  In  New  York  city  there  are  usually  fifteen 
to  thirty  cases  following  every  fourth  of  July.  Most  of 
them  are  caused  by  infection  through  blank  cartridge 
wounds.  Recovery  sometimes  follows  from  the  ordi- 
nary symptomatic  treatment  or  without  treatment  at 
all,  so  that  the  statistics  of  cures  of  the  disease  by  the 
injection  of  antitoxic  serum  must  be  very  carefully 
sifted  before  they  can  be  accepted  as  reliable.  Lambert, 
however,  who  has  recently  made  an  exhaustive  study 
of  tetanus,  states  that  in  a  total  of  114  cases  of  this 
disease  treated  with  antitoxin,  according  to  published 
and  unpublished  reports,  there  was  a  mortality  of  40.35 
per  cent.  Of  these,  47  were  acute  cases — that  is,  cases 
with  an  incubation  period  of  eight  days  or  less  and  with 
rapid  onset,  or  cases  with  a  longer  period  of  incubation, 
but  intensely  rapid  onset  of  symptoms;  of  these  the 
mortality  was  74.46  per  cent.  Of  the  chronic  type — 
those  with  an  incubation  period  of  nine  days  or  more, 
or  those  with  shorter  incubation  with  slow  onset — there 
were  61  cases,  with  a  mortality  of  1 6.39  per  cent.  With 
a  still  larger  number  of  cases  the  results  indicate  that 
with  tetanus  antitoxin  about  20  per  cent,  better  results 
are  obtained  than  without.  The  new  method  of  inject- 
ing from  3-15  c.c.  of  antitoxic  serum  into  the  lateral 
ventricles  has  not,  in  the  writer's  opinion,  shown  itself 
to  be  superior  to  the  intravenous  or  subcutaneous 


400  -BA  CTERIOL  OGY. 

methods.  Some  speak  well  of  it.  No  bad  results  have 
followed  the  injections  when  the  serum  was  sterile  and 
the  operation  was  performed  aseptically. 

The  Dosage  of  Tetanus  Antitoxin.  For  immunization 
10  c.c.  of  a  serum  of  a  strength  of  1:1,000,000,000 
will  suffice  unless  the  danger  seems  great,  when  the 
injection  is  repeated  at  the  end  of  a  week.  For  treat- 
ment, it  is  well  to  begin  with  50  c.c.,  and  then,  accord- 
ing to  the  severity  of  the  case,  give  from  20  to  50  c.c. 
each  day  until  the  symptoms  abate.  In  the  gravest 
cases  no  curative  effect  will  be  noticed  from  the  serum. 

Though  these  few  cases  are  not  sufficient  to  form  a 
final  judgment  of  any  treatment,  Lambert  concludes 
that  by  means  of  the  antitoxin  treatment,  combined 
with  other  rational  methods,  the  prognosis,  even  in 
acute  cases  of  tetanus,  has  been  improved;  but  that 
it  still  remains  exceedingly  grave — so  much  so  that 
the  preventive  inoculation  of  serum  in  all  cases  where 
dirt  has  been  ground  into  serious  contusions  de- 
serves a  much  more  extensive  consideration  than  has 
heretofore  been  given  it.  The  striking  results  which 
have  been  obtained,  particularly  in  veterinary  practice, 
with  the  prophylactic  injection  of  tetanus  antitoxin, 
would  seem  to  warrant  the  treating  of  patients  with 
immunizing  doses  of  serum — at  least  in  neighborhoods 
where  tetanus  is  not  uncommon — when  the  lacerated 
and  dirty  condition  of  their  wounds  may  indicate  the 
possibility  of  a  tetanus  infection. 

Differential  Diagnosis.  The  differential  diagnosis  of 
the  bacillus  of  tetanus  is,  generally  speaking,  not  diffi- 
cult, inasmuch  as  animal  inoculation  affords  a  sure  test 
of  the  specific  organism.  No  other  micro-organism 
known  produces  similar  effects  to  the  tetanus  bacillus, 


THE  BACILLUS  OF  TETANUS.  4Q1 

nor  is  any  other  neutralized  by  tetanus  antitoxin.  The 
other  characteristics  also  of  this  bacillus  are  usually  dis- 
tinctive, though  microscopical  examination  alone  cannot 
be  depended  on  to  make  a  differential  diagnosis.  Diffi- 
culty arises  when  other  anaerobic  or  aerobic  bacilli, 
almost  morphologically  identical  with  the  tetanus 
bacillus,  are  encountered  which  are  non-pathogenic, 
such  as  the  bacillus  pseudotetanicus  anaerobius,  already 
mentioned,  and  the  bacillus  pseudotetanicus  aerobius. 
It  is  possible,  however,  that  both  these  bacilli,  when 
characteristic  in  cultures,  are  only  varieties  of  the 
tetanus  bacillus,  which,  under  unfavorable  conditions  of 
growth,  have  lost,  their  virulence.  These  non-virulent 
types  do  not,  as  a  rule,  have  spores  absolutely  at  their 
ends,  and  the  spores  themselves  are  usually  more  ovoid 
than  those  in  the  true  tetanus  bacilli. 


26 


CHAPTER  XXIII. 

BACILLUS  TYPHOSUS  (EBERTH-GAFFKY?S  BACILLUS  OF 
TYPHOID   FEVER  ;   BACILLUS  TYPHI  ABDOMINALIS). 

THIS  organism  was  first  observed  by  Eberth,  and  inde- 
pendently by  Koch,  in  1880,  in  the  spleen  and  diseased 
organs  of  the  intestine  in  typhoid  cadavers,  but  was 
not  obtained  in  pure  culture  and  its  principal  biologi- 
cal cultures  described  until  the  researches  of  Gaffky, 
in  1884.  Its  etiological  relationship  to  typhoid  fever 
has  been  particularly  difficult  of  demonstration,  for 
although  pathogenic  for  many  animals  when  subcuta- 
neously  or  intravenously  inoculated,  it  has  been  almost 
impossible  to  produce  infection  or  in  any  way  give  rise 
to  lesions  corresponding  to  those  occurring  generally  in 
man.  It  has  been  recently  shown,  however,  that 
animals  under  certain  conditions,  when  their  power 
of  resistance  has  been  reduced,  as  by  exposure  to  the 
influence  of  noxious  gases,  may  be  rendered  susceptible 
to  infection,  with  the  production  of  more  or  less  char- 
acteristic lesions.  These  results,  together  with  the 
specific  reactions  of  the  blood-serum  of  typhoid  patients, 
as  first  pointed  out  by  Pfeiffer,  Gruber,  Widal,  and 
others,  and  the  constant  presence  of  the  bacillus 
typhosus  in  the  intestines  and  in  some  of  the  organs 
of  the  typhoid  cadavers,  as  shown  by  its  frequent 
isolation  from  the  spleen,  blood,  and  excretions  of  the 
sick  during  life  and  its  absence  in  healthy  persons, 


BACILLUS  TYPHOSUS. 


403 


unless  they  are  convalescent  from  typhoid  infection, 
have  demonstrated,  on  a  scientific  basis,  that  this  bacil- 
lus is  the  chief  etiological  factor  in  the  production  of 
typhoid  fever. 

Morphological  Characters.  The  typhoid  bacilli  are 
rods  of  about  l/i  to  3/2  in  length  by  0.5//  to  0  Sfjt  in 
diameter,  with  rounded  ends,  often  growing  into  long 
threads.  They  are  usually  longer  and  somewhat  more 
slender  in  form  than  the  bacilli  coli  communis  under 
similar  conditions.  The  typhoid  bacilli  vary,  however, 
in  shape  when  grown  in  different  culture  media.  (See 
Figs.  49,  50,  and  Fig.  6,  page  39.) 


FIG.  49. 


FIG.  50. 


Typhoid  bacilli  from  nutrient  agar. 
X  1100  diameters. 


Typhoid  bacilli  from  nutrient  gelatin. 
X  1100  diameters. 


The  typhoid  bacilli  stain  with  the  ordinary  aniline 
colors,  but  a  little  less  readily  than  do  most  other  bac- 
teria, though  there  is  no  constant  difference  in  staining 
characteristics  between  these  and  other  bacilli  of  this 
group — the  colon  bacilli.  They  are  decolorized  by 
Gram's  iodine  solution.  Not  infrequently,  particu- 
larly when  grown  on  potato,  refractive  granules  may 


404  BACTERIOLOGY. 

be  seen  at  the  ends  of  the  rods,  which  stain  more  in- 
tensely, and  either  at  the  extremities  or  along  the  body 
"  vacuoles"  are  observed,  which  remain  unstained;  but 
as  these  show  even  less  resistant  power  than  the  homo- 
geneous bacilli  found  in  other  cultures,  they  are  cer- 
tainly not  spores,  but  probably  are  evidences  only  of 
retrograde  changes  and  effects  of  the  drying  prepara- 
tory to  staining. 

FIG.  51. 


Flagella,  heavily  stained,  attached  to  bacilli. 

The  bacilli,  when  existing  under  favorable  conditions, 
are,  although  in  various  cultures  to  a  different  degree, 
very  actively  motile,  the  smaller  ones  having  often  an 
undulating  motion,  while  the  larger  rods  dart  about 
rapidly,  with  a  snake-like  movement.  This  movement 
is  produced  by  a  number  of  delicate  locomotive  organs 
in  the  form  of  fine,  hair-like  flagella,  which  are  arranged 
around  the  bodies  of  the  bacilli.  (Fig.  10,  page  43, 
and  Fig.  51.)  The  flagella  are  usually  from  eighteen 
to  twenty  in  number,  but  many  short  rods  have  but  a 
single  terminal  flagellum.  They  are  not  seen  in  un- 
stained preparations,  nor  are  they  rendered  visible  by 


BACILLUS  TYPHOSUS.  405 

the  ordinary  methods  of  staining.  (See  Staining  of 
Flagella,  page  205.) 

Biological  Characters.  The  typhoid  bacillus  is  a 
motile,  aerobic,  non-liquefying  bacillus,  developing 
best  at  37°  C.;  over  40°  and  below  30°  its  growth  is 
retarded;  below  10°  it  ceases.  It  grows  most  abun- 
dantly in  the  presence  of  oxygen,  but  oxygen  is  not 
essential  to  its  development. 

Its  growth  on  most  culture  media  is  similar  to  that 
of  the  bacillus  coli  conimunis,  but  it  is  somewhat 
slower  and  not  quite  so  luxuriant. 

FIG.  52. 


A  superficial  and  a  deep  colony  of  typhoid  bacilli  in  gelatin. 
X  50  diameters. 

Growth  on  Gelatin  Plates.  (Fig.  52.)  The  colonies 
growing  deep  down  in  this  plate  medium  have  nothing 
in  their  appearance  to  distinguish  them;  they  appear 
as  round  points  with  a -sharp  margin,  of  a  yellowish- 


406  BACTERIOLOGY. 

brown  color,  and  finely  granular.  The  superficial 
colonies,  however,  particularly  when  young,  are  often 
quite  characteristic;  they  are  transparent,  bluish-white 
in  color,  with  an  irregular  outline,  not  unlike  a  grape- 
leaf  in  shape.  Slightly  magnified  they  appear  homo- 
geneous in  structure,  but  marked  by  a  delicate  network 
of  furrows. 

In  stick  cultures  in  gelatin  the  growth  is  mostly  on  the 
surface,  appearing  as  a  thin,  scalloped  extension,  which 
gradually  reaches  out  to  the  sides  of  the  tube.  In  the 
track  of  the  needle  there  is  but  a  limited  growth,  which 
may  be  streaked,  granular,  or  uniform  in  structure,  and 
of  a  yellowish-brown  color.  There  is  no  liquefaction. 

Growth  in  Bouillon.  This  medium  is  uniformly 
clouded  by  the  typhoid  bacillus,  but  the  clouding  is  not 
so  intense  as  by  the  colon  bacillus.  A  film  is  frequently 
formed  on  the  surface  after  eighteen  to  twenty-four 
hours'  growth.  A  very  slight  amount  of  acid  is  pro- 
duced. 

Growth  on  Agar.  The  streak  cultures  on  agar  are 
not  distinctive;  a  transparent,  grayish  streak  is  formed. 

Growth  on  Potato.  The  growth  on  this  medium 
has  been  held  by  some  to  be  very  important,  but  it 
varies  considerably.  When  characteristic  the  growth 
is  invisible,  but  luxuriant,  usually  covering  the  surface 
of  the  medium,  and  when  scraped  with  the  needle  offers 
a  certain  resistance.  In  some  cases,  however,  the 
growth  is  restricted  to  the  immediate  vicinity  of  the 
point  of  inoculation,  not  very  luxuriant,  and  of  the 
same  color  as  the  potato.  Again,  the  growth  may  be 
quite  heavy  and  colojred  yellowish-brown,  and  with  a 
greenish  halo,  when  it  is  very  similar  to  the  growth  of 
the  colon  bacillus.  These  differences  of  growth  on  this 


BACILLUS  TYPHOSUS.  407 

medium  appear  to  be  chiefly  due  to  variations  in  the 
substance,  especially  in  the  reaction,  of  the  potato. 

Milk.  The  typhoid  bacillus  does  not  cause  coagu- 
lation when  grown  in  sterilized  milk. 

Fermentation.  It  does  not  produce  fermentation  in 
either  glucose,  lactose,  saccharose,  or  glycerin  bouillon, 
and  evolves  no  gas  as  the  result  of  fermentation. 

Lactose-litmus  Agar.  It  grows  usually  as  pale  blue 
colonies  on  lactose-litmus  agar,  but  occasionally  causes 
slight  reddening  of  the  surrounding  medium. 

Indol  Reaction.  It  does  not  produce  indol.  This 
test  was  proposed  by  Kitasato  for  differentiating  the 
typhoid  bacillus  from  other  similar  bacilli,  such  as 
those  of  the  colon  group,  which,  as  a  rule,  give  the 
indol  reaction. 

The  reaction,  being  a  very  delicate  one,  requires 
great  care  in  its  performance  to  arrive  at  accurate  con- 
clusions. (For  test  of  indol,  see  page  77.)  Instead  of 
bouillon,  the  simple  peptone-water  (which  consists  of 
dried  peptone,  1  part;  sodium  chloride,  0.5  part,  and 
distilled  water,  100  parts)  is  to  be  preferred  for  this 
purpose,  because  its  pale  color  does  not  mask  the  reac- 
tion*. 

Pathogenic  Properties.  It  has  been  extremely  diffi- 
cult to  show  experimentally  that  the  bacillus  typhosus 
is  specifically  pathogenic  for  animals.  A  great  many 
experiments  have  been  made,  with  the  view  of  repro- 
ducing in  the  tissues  of  lower  animals  the  pathological 
lesions  of  typhoid  fever  as  seen  in  man,  but  the  results 
have  not  been  completely  satisfactory  ;  nor  is  this  sur- 
prising when  one  considers  that  this  disease  does  not 
occur  naturally,  so  far  as  is  known,  among  animals. 
Sickness  or  fatal  results  without  the  appearance  of  the 


408  BACTERIOLOGY. 

typical  pathological  changes  have  regularly  followed 
animal  inoculations,  but  in  most  cases  they  could  easily 
be  traced  to  the  toxaemia  produced  by  the  substances  in 
the  bodies  of  the  bacilli  injected,  not  necessarily  accom- 
panied by  the  growth  of  the  organism,  rather  than  to 
infection  due  to  the  development  of  the  typhoid  bacillus 
in  the  tissues. 

In  a  certain  number  of  cases  subcutaneous  and  intra- 
peritoneal  inoculations  in  animals  have  been  productive 
of  more  or  less  typical  typhoid  lesions.  Among  the 
most  successful  efforts  in  this  direction  are  the  experi- 
ments of  Cygnaeus  and  Seitz,  who,  by  the  inoculation 
of  the  typhoid  bacillus  into  dogs,  rabbits,  and  mice, 
produced  in  the  small  intestines  conditions  that  were 
histologically  and  to  the  naked  eye  analogous  to  those 
found  in  the  human  subject,  but  their  results  were  not 
constant.  Of  a  number  of  experiments  made  by  Ab- 
bott, with  the  same  object  in  view,  only  one  positive 
result  followed  the  introduction  of  typhoid  bacilli  into 
the  circulation  of  rabbits.  In  this  case  the  ulcer  in 
the  ileum  was  macroscopically  and  microscopically  iden- 
tical with  those  found  at  autopsy  in  the  small  intestines 
of  the  human  subject  dead  of  this  disease.  The  bacilli 
were  found  in  the  spleen. 

Experiments  indicate  that  the  presence  of  other  bac- 
teria in  the  body,  and  of  exposure  to  the  effect  of  nox- 
ious gases  in  lowering  the  natural  resistance  of  the 
individual,  render  him  more  susceptible  to  infection 
from  typhoid  fever  and,  indeed,  from  other  infectious 
diseases. 

But  whatever  conclusions  may  be  drawn  from  these 
results,  with  regard  to  the  typhoid  process  in  animals, 
typhoid  fever  in  the  human  subject  is  now  recognized 


BACILLUS  TYPHOSUS.  409 

as  a  true  infection,  caused  by  the  introduction  and 
growth  of  typhoid  bacilli.  It  belongs  to  that  class  of 
infectious  diseases  which  are  known  as  metastatic — that 
is  to  say,  diseases  in  which  the  specific  bacilli  do  not 
abound  in  the  entire  circulation,  as  in  septicaemia,  nor 
remain  localized  in  one  place,  but  are  distributed  in 
groups  throughout  the  body.  The  characteristic  lesions 
of  typhoid  fever  are  seated  in  the  lymphatic  structures  of 
the  intestine — namely,  the  solitary  follicles  and  patches 
of  Peyer,  the  mesenteric  glands,  and  the  spleen.  The 
liver  and  kidneys  are  less  commonly  attacked.  Wher- 
ever found  the  typhoid  bacilli  are  observed  to  be  arranged 
in  groups  or  foci;  only  occasionally,  as  in  the  walls  of 
the  intestine,  are  they  singly  or  loosely  aggregated  to- 
gether. These  foci  are  formed,  most  probably,  during 
life,  as  is  proved  by  the  degenerative  changes  often 
seen  about  them;  but  it  is  possible  that  the  bacilli  may 
also  multiply  somewhat  after  death. 

The  production  of  the  lymph-nodules  so  often  found 
in  typhoid  fever  in  the  internal  organs  is  due  to  the 
effects  of  the  toxic  substances  eliminated  by  the  typhoid 
bacilli.  This  hyperplasia  is  particularly  evident  in  the 
lymphatic  structures  of  the  intestine,  these  being  more 
directly  under  the  influence  of  the  concentrated  products 
of  the  bacilli.  To  these,  however,  other  inflammatory 
processes  are  added,  until  finally  necrosis  or  sloughing 
of  the  tissues  takes  place.  Possibly  all  these  series  of 
changes  may  be  at  times  caused  solely  by  the  products 
of  the  typhoid  bacilli  which  are  gathered  at  certain 
points.  There  is  no  question,  however,  that  usually 
other  organisms  take  part  in  the  production  of  these 
processes  in  the  intestines,  but  it  remains  to  be  deter- 
mined when  they  begin  to  do  so.  In  typhoid  fever 


410  BACTERIOLOGY. 

necrosis  of  the  tissues  of  the  internal  organs  is  of 
comparatively  rare  occurrence.  Caseation  of  the  mes- 
enteric  glands,  which  is  commonly  observed,  is  due 
probably  to  mixed  infection.  There  are,  however,  a 
number  of  cases  now  on  record  in  which  the  typhoid 
bacillus  has  played  the  part  of  pus  producer.  Cases 
of  sacculated  and  general  peritonitis,  subphrenic  ab- 
scess, osteomyelitis,  periostitis,  and  inflammatory  pro- 
cesses of  other  kinds  have  been  reported  as  being 
due  to  the  typhoid  bacillus.  Kruse  also  reports  an 
abscess  of  the  spleen  which  contained  only  bacillus 
typhosus,  and  typhoid  abscess  of  the  liver  has  been 
recorded  by  many.  In  certain  cases  of  typhoid  pneu- 
monia, serous  pleurisy,  empyema,  and  meningitis, 
typhoid  bacilli  exclusively  have  occurred.  The  in- 
flammation produced  may  or  may  not  be  accompanied 
by  the  formation  of  pus.  As  argument  against  the 
observations  above  cited  there  has  been  brought  forward 
the  supposition  that  probably  the  real  cause  of  the 
disease  had  been  destroyed  before  the  entrance  of  the 
typhoid  bacillus.  Though  this  may  be  true  of  some 
cases,  as  in  pneumonia,  which  is  caused  usually  by  the 
short-lived  pneumococcus,  there  is  no  reason  to  doubt 
the  causal  relation  of  the  typhoid  bacillus  to  the  other 
diseases,  inasmuch  as  it  has  been  proved  by  numerous 
investigations. 

Such  cases,  however,  are  of  comparatively  rare  occur- 
rence, because  only  exceptionally  do  the  bacilli  suffi- 
ciently mass  together  in  such  numbers  as  to  become 
pus  producers.  As  a  rule,  when  complications  occur 
in  typhoid  fever  they  are  due  to  secondary  or  mixed  in- 
fection with  the  staphylococcus,  pneumococcus,  strepto- 
coccus, pyocyaneus,  and  colon  bacillus.  Frequently 


BACILLUS  TYPHOSUS.  411 

these  bacteria  are  found  side  by  side  with  the  typhoid 
bacilli;  in  such  cases  it  is  difficult  to  say  which  was  the 
primary  and  which  was  the  secondary  infection. 

The  peculiar  arrangement  of  the  typhoid  bacilli  in  the 
body  can  only  be  explained  by  their  passage  through 
the  circulation;  and  this  is  proved  by  the  bacilli  being 
found  in  the  spleen  almost  constantly  and  in  smaller 
numbers  in  the  blood  itself.  Thus,  Neuhauss  has  had 
nine  positive  results  out  of  fifteen  in  cultures  from  vein 
blood. 

The  typhoid  bacillus  can  be  transmitted  also  from 
the  blood  of  the  mother  to  the  foetus  (E berth,  Fraenkel, 
etc.).  In  one  case  reported  by  Ernst  a  living  child, 
four  days  after  birth,  showed  evidences  of  general 
typhoid  infection,  icterus  and  rose-spots.  Frascani 
reports  that  in  animal  experiments  he  has  frequently 
found  typhoid  bacilli  in  the  foetus. 

Not  infrequently  typhoid  bacilli  are  found  in  the 
secretions.  They  are  present  in  the  urine  in  about  20 
per  cent,  of  the  cases  in  the  third  and  fourth  week  of 
typhoid  fever.  Slight  pathological  lesions  in  the  kid- 
neys almost  always  occur  in  typhoid  fever,  but  severe 
lesions  also  sometimes  occur.  In  a  case  under  our  ob- 
servation the  urine  was  distinctly  purulent  and  crowded 
with  typhoid  bacilli.  The  bacillus  typhosus  is  not 
commonly  found  in  the  sweat,  but  Geisler  observed  it 
once.  It  has  also  been  detected,  though  rarely,  in  the 
sputum  and  secretions  of  the  throat. 

In  cases  of  pneumonia  due  to  the  typhoid  bacillus  it 
is  abundantly  present  in  the  sputa,  and  care  should  be 
taken  to  disinfect  the  expectoration  of  typhoid  patients. 
According  to  Chiari,  in  typhoid  fever  the  bacilli  are 
almost  always  present  in  the  gall-bladder.  The  bacilli 


412  BACTERIOLOGY. 

are  frequently  eliminated  by  the  feces  being  derived 
from  the  inflamed^  mucous  surface  of  the  intestines; 
their  growth  within  the  intestinal  canal  itself,  even  if 
it  occurs  to  a  limited  extent,  is  probably  not  extensive. 
Methods  of  Infection.  With  regard  to  the  mode  of 
invasion  of  the  typhoid  bacilli,  there  is  no  doubt  that 
it  is  principally  by  way  of  the  mouth,  through  the 
stomach  to  the  intestines.  Mayer  reports  a  particularly 
convincing  illustration  of  this  fact  in  a  case  where  death 
ensued  on  the  second  day  of  the  disease.  On  autopsy 
were  found  hypersemia  of  the  lungs,  spleen,  and  kid- 
ney; in  the  lower  portion  of  the  ileum  great  enlarge- 
ment of  the  solitary  follicles  and  patches  of  Peyer,  but 
nowhere  a  trace  of  necrosis  or  loss  of  substance;  nor 
were  the  mesenteric  glands  enlarged.  Microscopically 
an  extraordinary  deposit  of  characteristic  bacilli  were 
found  in  the  submucosa  and  interstitial  spaces  of  the 
muscles;  many  hundred  bacilli  lay  in  one  field.  On 
the  other  hand,  several  cases  are  recorded  in  which  the 
intestinal  changes  were  entirely  wanting,  and  only  a 
localization  of  bacilli  and  lesions  in  the  mesenteric 
glands  and  spleen  revealed  the  nature  of  the  infection. 
Inasmuch  as  they  were  present  in  the  lymph-glands 
which  belong  to  the  intestines,  it  may  be  assumed, 
thinks  Kruse,  who  reports  one  of  these  cases,  that  the 
bacilli  were  here  more  rapidly  absorbed  than  usual  with- 
out multiplying  to  any  extent  in  the  intestines.  The 
case  mentioned  by  Guarnieri  is  also  worthy  of  notice: 
in  this  there  was  apparently  a  primary  infection  of  the 
gall-ducts,  with  no  accompanying  lesions  in  the  intes- 
tine. Bacilli  were  found  in  the  blood  twelve  days  be- 
fore death,  and  on  autopsy  pure  cultures  were  obtained 
from  the  liver  and  spleen. 


BACILLUS  TYPHOSUS.  413 

Not  only  do  the  very  great  majority  of  cases  exam- 
ined bacteriologically  and  pathologically,  but  the  epi- 
demiological  history  of  the  disease,  prove  that  the 
chief  mode  of  invasion  of  the  typhoid  bacillus  is  by 
way  of  the  mouth  and  stomach.  The  infective  mate- 
rial is  discharged  principally  by  means  of  the  excretions 
and  secretions  of. the  sick — namely,  by  the  feces,  the 
urine,  and  occasionally  by  the  sputum. 

Of  considerable  practical  importance  is  it  to  know  for 
what  length  of  time  the  typhoid  bacillus  is  capable  of 
living  outside  of  the  body;  but,  unfortunately,  owing 
to  the  great  difficulties  in  proving  the  presence  of  this 
organism  in  natural  conditions,  our  knowledge  on  this 
point  is  very  deficient.  In  feces  the  length  of  life  of 
the  typhoid  bacilli  is  very  variable;  sometimes  they 
live  but  a  few  hours,  usually  a  few  days,  exceptionally 
for  very  long  periods.  Thus,  according  to  Uffelmann, 
typhoid  bacilli  may  remain  alive  in  feces  for  five  and 
a  half  months,  and,  according  to  Karlinski,  for  at  least 
several  months.  Foote  says  that  they  can  be  found  in 
living  oysters  for  a  month  at  a  time.  Their  life  in 
feces  and  in  water,  however,  is  usually  very  much 
shorter.  As  a  rule,  they  can  be  detected  in  water  no 
longer  than  fourteen  days  after  introduction.  The 
life  of  the  typhoid  bacillus  varies  according  to  the 
abundance  and  varieties  of  the  bacteria  associated  with 
it  and  according  to  the  presence  or  absence  of  such  in- 
jurious influences  as  high  temperature,  light,  desicca- 
tion, etc.,  to  which  it  is  peculiarly  sensitive.  That  the 
bacilli  do  live  much  longer  under  favorable  circum- 
stances, as  to  protection  and  nourishment,  than  is  gen- 
erally supposed,  is  shown  by  the  fact,  as  reported  by 
Buschke,  that  they  were  found  in  an  old  bone-centre 


414  BACTERIOLOGY. 

seven  years  after  the  original  infection.  There  is  no 
reason  to  deny  that  such  opportunities  for  a  latent  ex- 
istence of  the  typhoid  bacillus  may  not  occur  outside 
of  the  body.  Indeed,  many  epidemics  of  typhoid 
fever  can  only  be  accounted  for  by  some  such  assump- 
tion of  latency  in  or  outside  of  the  body. 

The  bacilli  may  reach  the  mouth  by  means  of  infected 
fingers  or  articles  of  various  kinds,  or  by  the  ingestion 
of  infected  food,  milk,  water,  etc.,  or  by  more  obscure 
ways,  such  as  the  contamination  of  food  by  flies  and 
other  insects,  or  by  the  inhalation  through  the  mouth 
of  dust  containing  typhoid  bacilli.  Of  the  greatest 
importance,  however,  is  the  production  of  infection 
by  contaminated  drinking-water  or  through  drinking- 
water  or  milk,  which  is  the  most  plausible  explana- 
tion for  the  majority  of  epidemics  of  typhoid  fever. 
In  many  cases  indirect  proof  of  this  mode  of  infec- 
tion has  been  found  in  the  known  contamination  of 
the  water  with  typhoid  feces  or  urine,  and  in  some  few 
cases  it  has  been  confirmed  by  direct  proof  in  finding 
the  bacilli.  Examples  of  infection  from  water  and 
milk  have  come  frequently  under  our  direct  observa- 
tion— for  instance,  a  large  force  of  workmen  obtained 
their  drinking-water  from  a  well  very  near  to  their 
work.  Typhoid  fever  broke  out,  and  continued  to 
spread  until  the  well  was  filled  up.  Investigation 
showed  that  some  of  the  sick,  before  their  discovery, 
repeatedly  infected  the  soil  surrounding  the  well  with 
their  urine  and  feces.  Another  instance  of  milk  in- 
fection secondary  to  water  infection  was  the  case  of  a 
milk  dealer  whose  son  came  home  suffering  from 
typhoid  fever.  The  intestinal  movements  were  thrown 
into  a  small  stream  which  ran  into  a  pond  from  which 


BACILLUS  TYPHOSUS.  415 

the  milk  cans  were  washed.  A  very  alarming  epidemic 
of  typhoid  developed,  which  was  confined  to  the  houses 
and  asylums  supplied  with  this  milk.  In  our  late  war, 
not  only  water  infection  but  food  infection  was  notice- 
able, as  in  the  case  of  a  regiment  where  certain  com- 
panies were  badly  infected,  while  others  nearly  escaped. 
Each  company  had  its  separate  kitchen  and  food-supply, 
and  much  of  the  infection  could  be  traced  to  the  food. 

In  this,  as  in  all  infectious  diseases,  individual  sus- 
ceptibility plays  an  important  role  in  the  production  of 
infection.  Without  a  suitable  soil  upon  which  to  grow 
the  seed  cannot  thrive.  There  must  in  many  be  some 
disturbance  of  the  digestion,  excesses  in  drinking,  etc., 
or  a  general  weakening  of  the  power  of  resistance  of 
the  individual,  caused  by  bad  food,  exposure  to  heat, 
overexertion,  etc.,  as  with  soldiers  and  prisoners,  for 
example,  to  bring  about  the  conditions  suitable  for  the 
production  of  typhoid  fever. 

The  supposition  that  the  breathing  of  noxious  gases 
is  conducive  to  the  disease,  though  possibly  true  to  a 
certain  extent,  as  some  animal  experiments  already 
referred  to  would  seem  to  indicate,  has  not  yet  been 
conclusively  proven;  nor  do  Pettenkofer's  investiga- 
tions, into  the  relation  of  the  frequency  of  typhoid 
fever  to  the  ground-water  level,  satisfactorily  explain 
the  occurrence  of  the  disease  in  most  cases,  whether 
sporadically  or  in  epidemics. 

Immunization.  Specific  immunization  against  experi- 
mental typhoid  infection  has  been  produced  in  mice, 
guinea-pigs,  rabbits,  dogs  and  other  animals  by  the 
usual  method  of  injecting  at  first  small  quantities  of 
the  living  or  dead  typhoid  culture  and  gradually  in- 
creasing the  dose.  The  blood-serum  of  animals  thus 


416  BACTERIOLOGY. 

immunized  has  been  found  to  acquire  protective  and 
curative  bactericidal  and  perhaps  feeble  antitoxic  prop- 
erties against  the  typhoid  bacillus.  These  character- 
istics have  also  been  observed  in  the  blood-serum  of 
persons  who  are  convalescent  from  typhoid  fever 
(Pfeiffer  and  Kolle,  Widal  and  Chantemesse).  Re- 
cently the  attempt  has  been  made  to  employ  the 
typhoid-serum  for  the  cure  of  typhoid  fever  in  man, 
but  no  marked  results  have  been  obtained.  The  injec- 
tion in  man  of  very  small  amounts  (0.3  c.c.  of  bouillon 
culture)  of  dead  typhoid  bacilli  produces  for  a  day  or 
two  a  slight  fever  reaction,  to  be  followed  in  a  few  days 
by  the  development  of  bactericidal  substances  in  the 
blood,  which  apparently  are  sufficient  in  amount  to 
give  immunity  for  some  weeks.  The  use  of  immu- 
nized serum,  or  when  this  cannot  be  obtained  of  dead 
cultures,  would  seem  to  be  advisable  where  great 
danger  of  typhoid  infection  exists. 

The  Diagnosis  of  Typhoid  Fever,  or  rather  of  Typhoid 
Infection,  by  Means  of  the  Widal  or  Serum  Reaction. 
The  chief  practical  application  of  our  knowledge  of  the 
specific  substances  developed  in  the  blood  of  persons 
sick  with  typhoid  fever  has  been  in  the  way  of 
diagnosis.  In  view  of  the  interest  which  has  been 
manifested  in  this  test,  and  of  the  fact  that  it  is  now 
so  largely  used,  a  brief  history  of  the  investigations 
which  led  up  to  its  discovery  may  be  given. 

In  1894-95,  Pfeiffer  showed  that  when  cultures  con- 
taining dead  or  living  cholera  spirilla  or  typhoid  bacilli 
are  injected  subcutaneously  into  animals  or  man,  specific 
protective  substances  are  formed  in  the  blood  of  the  in- 
dividuals thus  treated.  These  substances  grant  a  more 
or  less  complete  immunity  against  the  invasion  of  the 


BACILLUS  TYPHOSUS.  417 

living  germs  of  the  respective  diseases.  He  also  de- 
scribed the  occurrence  of  a  peculiar  phenomenon  when 
a  portion  of  a  fresh  culture  of  the  typhoid  bacillus  on 
agar  is  added  to  a  small  quantity  of  the  serum  of  an 
animal  immunized  against  typhoid  and  the  mixture 
injected  into  the  peritoneal  cavity  of  a  non-immunized 
guinea-pig.  After  this  procedure,  if  from  time  to  time 
minute  drops  of  the  liquid  be  withdrawn  in  a  capillary 
tube  and  examined  microscopically,  it  is  found  that  the 
bacteria,  which  were  formerly  and  in  control  animals, 
which  remain,  actively  motile  and  vigorous,  become 
in  a  very  short  time,  under  the  influence  of  the  serum, 
entirely  motionless  and  later  dead.  They  are  first  im- 
mobilized, then  they  become  somewhat  swollen  and 
agglomerated  into  balls  or  clumps,  which  gradually 
become  paler  and  paler,  until  finally  they  are  dissolved 
in  the  peritoneal  fluid.  This  process  takes  place  regu- 
larly in  about  twenty  minutes,  provided  a  sufficient 
degree  of  immunity  be  present  in  the  animals  from 
which  the  serum  was  obtained.  The  animals  injected 
with  the  mixture  of  the  serum  of  immunized  animals 
and  typhoid  cultures  remain  unaffected,  while  control 
animals  treated  with  a  fluid  containing  only  the  serum 
of  non-immunized  animals  mixed  with  typhoid  cultures 
die.  Pfeiffer  claimed  that  the  reaction  of  the  serum 
thus  employed  is  so  distinctly  specific  that  it  may  serve 
for  the  differential  diagnosis  of  the  cholera  vibrion  or 
typhoid  bacillus  from  other  vibrions  or  allied  bacilli, 
such  as  Finkler's  and  Prior's  or  colon  groups. 

In  March,  1896,  Pfeiffer  and  Kolle  published  an 
article  entitled  "  The  Differential  Diagnosis  of  Typhoid 
Fever  by  Means  of  the  Serum  of  Animals  Immunized 
Against  Typhoid  Infection/ '  in  which  they  claimed 

27 


418  BACTERIOLOGY. 

that  by  the  aid  of  the  presence  or  absence  of  this  reac- 
tion in  the  serum  of  convalescents  from  suspected 
typhoid  fever  the  nature  of  the  disease  could  be  deter- 
mined. It  was  further  found  if  the  serum  of  an  animal 
thoroughly  immunized  to  the  typhoid  bacillus  was 
diluted  with  40  parts  of  bouillon,  and  a  similar  dilu- 
tion made  of  the  serum  of  non-immunized  animals, 
and  both  solutions  were  then  inoculated  with  a  culture 
of  the  typhoid  bacillus  and  placed  in  the  incubator  at 
37°  C.,  that  after  the  expiration  of  one  hour  macro- 
scopical  differences  in  the  culture  could  be  observed, 
which  increased  in  distinctness  for  four  hours  and  then 
gradually  disappeared.  The  reaction  occurring  is  de- 
scribed as  follows  :  In  the  tubes  in  which  the  typhoid 
culture  is  mixed  with  typhoid  serum  the  bacilli  are 
agglomerated  in  fine,  whitish  flakes,  which  settle  to  the 
bottom  of  the  tube,  while  the  supernatant  fluid  is  clear 
or  only  slightly  cloudy.  On  the  other  hand,  the  tubes 
containing  mixtures  of  bouillon  with  cholera  or  coli 
serum,  or  the  serum  of  non-immunized  animals  inocu- 
lated with  the  typhoid  bacilli,  became  and  remained 
uniformly  and  intensely  cloudy.  These  serum  mix- 
tures, examined  microscopically  in  a  hanging  drop, 
show  distinct  differences.  The  typhoid  serum  mixture 
inoculated  with  the  typhoid  bacilli  exhibits  the  organ- 
isms entirely  motionless,  lying  clumped  together  in 
heaps;  in  the  other  mixtures  the  bacilli  are  actively 
motile. 

These  observations  were  made  independently  by 
Gruber  and  Durham,  who  maintained,  however,  that 
the  reaction  described  by  Pfeiffer  was  by  no  means 
specific,  and  that  when  the  reaction  is  positive  the 
diagnosis  still  remains  in  doubt,  for  the  reaction  is 


BA  CILL  US  TYPHOS  US.  419 

quantitative  only,  and  not  qualitative,  so  far  as  the 
cholera  spirillum  and  typhoid  bacillus,  at  least,  are 
concerned.  They  conclude,  nevertheless,  that  these 
investigations  will  render  valuable  assistance  in  the 
clinical  diagnosis  of  cholera  and  typhoid  fever.  It 
developed  through  further  research  that  before  the 
development  of  the  bactericidal  substances  agglutina- 
tive substances  usually  appeared  in  the  blood. 

WIDAI^XES^  The  first  practical  application  of  the 
use  Trf^serum,  however,  for  the  early  diagnosis  of 
typhoid  fever  on  a  more  extensive  scale  was  made  by 
Widal,  and  reported  with  great  fulness  and  detail  in  a 
communication  published  in  June,  1896.  Widal  con- 
firmed the  reaction  as  above  described,  proved  that  the 
agglutinative  reaction  was  one  of  infection  and  usually 
occurred  early,  elaborated  the  test,  and  proposed  a 
method  by  which  it  may  be  practically  applied  for 
diagnostic  purposes.  Since  then  the  serum  test  for 
the  diagnosis  of  typhoid  fever  has  come  into  general 
use  in  bacteriological  laboratories  in  all  parts  of  the 
world,  and  though  the  extravagant  expectations  raised 
at  the  time  when  Widal  first  announced  his  method  of 
applying  this  test  have  not  been  entirely  fulfilled,  it 
has,  nevertheless,  proved  to  be  of  great  assistance  in 
the  diagnosis  of  obscure  cases  of  the  disease,  and  it  is 
now  one  of  the  recognized  tests  for  the  differentiation 
of  the  typhoid  bacillus. 

It  should  also  be  mentioned  that  to  Wyatt  Johnson, 
of  Montreal,  belongs  the  credit  of  having  brought  this 
test  more  conspicuously  before  the  public  by  intro- 
ducing its  use  into  municipal  laboratories,  suggesting 
that  dried  blood  should  be  employed  in  place  of  blood- 
serum  (Widal  having  previously  noticed  that  drying 


420  BACTERIOLOGY. 

did  not  destroy  the  agglutinating  properties  of  typhoid 
blood);  and  that  in  October,  1896,  the  serum  test  was 
regularly  employed  in  the  New  York  Board  of  Health 
Laboratory  for  the  routine  examination  of  the  blood- 
serum  of  suspected  cases  of  typhod  fever.  Since  then 
numerous  health  departments  have  followed  the  example 
set  by  those  of  Montreal  and  New  York. 

USE  OF  DRIED  BLOOD.  Directions  for  Preparing 
Specimens  of  Blood.  The  skin  covering  the  tip  of  the 
finger  or  the  ear  is  thoroughly  cleansed,  and  is  then 
pricked  with  a  needle  deeply  enough  to  cause  several 
drops  of  blood  to  exude.  Two  fair-sized  drops  are  then 
placed  on  a  glass  slide,  one  near  either  end,  and  allowed 
to  dry.  Paper  may  also  be  employed,  but  it  is  not  as 
good,  for  the  blood  soaks  more  or  less  into  it,  and 
later,  when  it  is  dissolved,  some  of  the  paper-fibre  is 
apt  to  be  rubbed  off  with  it.  The  slide  is  placed  in  a 
box  for  protection. 

Preparation  of  Specimen  of  Blood  for  Examination. 
In  preparing  the  specimens  for  examination  the  dried 
blood  is  brought  into  solution  by  adding  to  it  and  mix- 
ing it  with  about  five  times  the  quantity  of  water  ;  then 
a  minute  drop  of  this  decidedly  reddish  mixture  is 
placed  on  a  cover-glass,  and  to  it  is  added  a  similar 
drop  of  an  eighteen  to  twenty-four-hour-old  bouillon 
culture  of  the  typhoid  bacillus,  which,  if  it  has  a  slight 
pellicle,  should  be  well  shaken.  The  drops,  after  being 
mixed,  should  have  a  faint  reddish  or  pink  tinge.  The 
cover-glass  with  the  mixture  on  the  surface  is  inverted 
over  a  hollow  slide  (the  edges  about  the  concavity 
having  been  smeared  with  vaseline,  so  as  to  make  a 
closed  chamber),  and  the  hanging  drop  then  examined 
under  the  microscope  (preferably  by  gaslight),  a  high- 


BACILLUS  TYPHOSUS. 


421 


power  dry  lens  (about  1/8  inch)  being  used,  or,  some- 
what less  serviceably,  a  1/12  oil-immersion  lens. 

THE  REACTION.  If  the  reaction  takes  place  rapidly 
the  first  glance  through  the  microscope  reveals  the  com- 
pleted reaction,  all  the  bacilli  being  in  loose  clumps 
and  nearly  or  altogether  motionless  (Fig.  53).  Be- 

FlG.  53. 


Widal  reaction.    Bacilli  gathered  into  one  large  and  two  small  clumps,  the 
few  isolated  bacteria  being  motionless  or  almost  so. 


tween  the  clumps  are  clear  spaces  containing  few  or  no 
isolated  bacilli.  If  the  reaction  is  a  little  less  complete 
a  few  bacilli  may  be  found  moving  slowly  between  the 
clumps  in  an  aimless  way,  while  others  attached  to  the 
clumps  by  one  end  are  apparently  trying  to  pull  away, 
much  as  a  fly  caught  on  fly-paper  struggles  for  freedom. 
If  the  agglutinating  substances  are  still  less  abundant 
the  reaction  may  be  watched  through  the  whole  course 
of  its  development.  Immediately  after  mixing  the  blood 
and  culture  together  it  will  be  noticed  that  the  bacilli 


422  BACTERIOLOGY. 

move  more  slowly  than  before  the  addition  of  serum. 
Some  of  these  soon  cease  all  progressive  movement, 
and  it  will  be  seen  that  they  are  gathering  together  in 
small  groups  of  two  or  more,  the  individual  bacilli 
being  still  somewhat  separated  from  each  other.  Grad- 
ually they  close  up  the  spaces  between  them,  and  clumps 
are  formed.  According  to  the  completeness  of  the 
reaction,  either  all  of  the  bacilli  may  finally  become 
clumped  and  immobilized  or  only  a  small  portion  of 
them,  the  rest  remaining  freely  motile,  and  those 
clumped  may  appear  to  be  struggling  for  freedom. 
With  blood  containing  a  large  amount  of  agglutinating 
substances  all  the  gradations  in  the  intensity  of  the  re- 
action may  be  observed,  from  those  shown  in  a  marked 
and  immediate  reaction  to  those  appearing  in  a  late  and 
indefinite  one,  by  simply  varying  the  proportions  of 
blood  added  to  the  culture  fluid. 

Pseudo-reactions.  If  too  concentrated  a  solution  of 
dried  blood  from  a  healthy  person  is  employed  there 
will  be  an  immobilization  of  the  bacilli,  but  no  true 
clumping.  This  is  sometimes  mistaken  for  a  reaction. 
Again,  dissolved  blood  always  shows  a  varying  amount 
of  detritus,  partly  in  the  form  of  fibrinous  clumps;  and 
prolonged  microscopical  examination  of  the  mixture  of 
dissolved  blood  with  a  culture  fluid  shows  that  the 
bacilli,  inhibited  by  substances  in  the  blood,  often  be- 
come more  or  less  entangled  in  these  clumps,  and  in 
the  course  of  one-half  to  one  hour  very  few  isolated 
motile  bacteria  are  seen.  The  fibrinous  clumps  alone, 
especially  if  examined  with  a  poor  light  by  a  beginner, 
may  be  easily  mistaken  for  clumps  of  bacilli.  Again, 
the  bacilli  may  become  clumped  after  remaining  for 
one-half  to  two  hours  by  slight  drying  of  the  drop  or 


BACILLUS  TYPHOSUS.  423 

the  effect  of  substances  on  the  cover-glass.  The  reac- 
tion in  typhoid  is  chiefly  due  to  specific  substances, 
but  clumping  and  inhibition  of  movement  similar  in 
character  may  be  caused  by  other  substances  such  as 
exist  in  normal  horse  and  other  serums.  This  is  a 
very  important  fact  to  keep  in  mind. 

USE  OF  SERUM.  Mode  of  Obtaining  Serum  for  Ex- 
amination. Fluid  blood-serum  can  be  easily  obtained  in 
two  ways:  First,  the  serum  may  be  obtained  directly  from 
the  blood,  thus  :  The  tip  of  the  finger  or  ear  is  pricked 
with  a  lancet-shaped  needle,  and  the  blood  as  it  issues 
is  allowed  to  fill  by  gravity  a  capillary  tube  having  a 
central  bulb.  The  ends  of  the  tube  are  then  sealed 
by  heat  or  wax,  and  as  the  blood  clots  a  few  drops  of 
serum  separate.  This  method  of  obtaining  blood- 
serum  has  the  advantage  of  rapidity;  but  it  has  also 
disadvantages — namely,  that  the  serum  thus  separated 
is  apt  to  contain  more  or  less  blood-cells,  which  some- 
what obscure  the  field  when  the  liquid  serum  is  imme- 
diately mixed  with  the  culture,  and  the  needle  stab  is 
often  objected  to.  Second,  the  serum  may  be  obtained 
from  blisters.  This  gives  more  satisfactory  results,  but 
causes  twelve  hours'  delay.  The  method  is  as  follows: 
A  section  of  cantharides  plaster,  the  size  of  a  5-cent 
piece,  is  applied  to  the  skin  at  some  spot  on  the  chest 
or  abdomen.  A  blister  forms  in  from  six  to  eighteen 
hours.  This  should  be  protected  from  injury  by  a 
vaccine  shield  or  bunion  plaster.  The  serum  from 
the  blister  is  collected  in  a  capillary  tube,  the  ends 
of  which  are  then  sealed.  Several  drops  of  the  serum 
can  be  easily  obtained  from  a  blister  so  small  that  it  is 
practically  painless  and  harmless.  The  serum  obtained 
is  clear  and  admirablv  suited  for  the  test. 


424  BACTERIOLOGY. 

Advantages  and  Disadvantage  of  Serum  and  Dried 
Blood  for  the  Serum  Test.  The  dried  blood  is  easily 
and  quickly  obtained,  and  does  not  deteriorate  or  be- 
come contaminated  by  bacterial  growth.  It  is  readily 
transported,  and  seems  to  be  of  nearly  equal  strength 
with  the  serum  in  its  agglutinating  properties.  It 
must  in  use,  however,  be  diluted  with  at  least  five 
times  its  bulk  of  water,  otherwise  it  is  too  viscid  to  be 
properly  employed.  The  amount  of  dilution  can  only 
be  determined  roughly  by  the  color  of  the  resulting 
mixture,  for  it  is  impossible  to  estimate  accurately  the 
amount  of  dried  blood  from  the  size  of  the  drop,  and 
it  is  too  much  trouble  to  weigh  it  accurately.  Serum, 
on  the  other  hand,  can  be  used  in  any  dilution  desired, 
varying  from  a  mixture  which  contains  equal  parts  of 
serum  and  broth  culture  to  that  containing  1  part  of 
serum  to  100  parts  of  culture,  and  this  can  be  exactly 
measured  by  a  graduated  pipette,  or,  roughly,  by  a 
measured  platinum  loop.  The  disadvantages  in  the 
use  of  serum  are  entirely  due  to  the  slight  difficulty  in 
collecting  and  transporting  it  and  the  delay  in  obtain- 
ing it  when  a  blister  is  employed.  If  the  serum  is 
obtained  from  blood  after  clotting  has  occurred  a 
greater  quantity  of  blood  must  be  drawn  than  is  neces- 
sary when  the  dried-blood  method  is  used  ;  if  it  is 
obtained  from  a  blister,  a  delay  of  six  to  eighteen 
hours  is  required.  The  transportation  of  the  serum 
in  capillary  tubes  presents  no  difficulties  if  tubes  of 
sufficiently  thick  and  tough  glass  are  employed  and 
placed  in  tiny  wooden  boxes.  For  scientific  investiga- 
tions and  for  accurate  results,  particularly  in  obscure 
cases,  the  use  of  fluid  serum  is  to  be  preferred  to  dried 
blood.  Practically,  however,  the  results  are  nearly  as 


BACILLUS  TYPHOSUS.  425 

good  for  diagnostic  purposes  from  the  dried  blood  as 
from  the  serum. 

The  Typhoid  Culture  Employed.  It  is  important  that 
the  culture  employed  for  serum-tests  should  be  a  suit- 
able one,  for  although  in  our  experience  all  cultures 
show  the  reaction,  yet  some  respond  much  better  than 
others.  A  broth  culture  of  the  typhoid  bacillus  de- 
veloped at  35°  C.,  not  over  twenty-four  hours  old,  in 
which  the  bacilli  are  isolated  and  actively  motile,  has 
been  found  to  give  us  the  most  satisfactory  results. 
Stock  cultures  of  typhoid  bacilli  can  be  preserved  on 
nutrient  agar  in  sealed  tubes  and  kept  in  the  ice-box. 
These  remain  alive  for  months  or  even  years.  From 
time  to  time  one  of  these  is  taken  out  and  used  to 
start  a  fresh  series  of  bouillon  cultures. 

The  Dilution  of  the  Blood-serum  to  be  Employed  and 
the  Time  Required  for  the  Development  of  Reaction.  The 
serum  test,  as  has  been  pointed  out,  is  quantitative  and 
not  qualitative.  By  this  it  is  not  meant  to  assert  that  the 
agglutinating  and  immobilizing  substances  produced  in 
the  blood  of  a  patient  suffering  from  typhoid  infection 
are  the  same  as  those  present  at  times  in  normal  blood, 
or  those  produced  in  the  blood  of  persons  sick  from 
other  infections.  It  is  intended,  however,  to  maintain 
that  the  effect  upon  the  bacilli,  as  seen  under  the  micro- 
scope, is  identical,  the  difference  being  that  in  typhoid 
fever,  as  a  rule,  substances  which  cause  this  reaction 
are  usually  far  in  excess  of  the  amount  which  ever 
appears  in  non  typhoid  blood,  so  that  the  reaction 
occurs  after  the  addition  to  the  culture  of  far  smaller 
quantities  of  serum  than  in  other  diseases,  or  when  the 
same  dilution  is  used  it  occurs  far  more  quickly  and 
completely  with  the  typhoid  serum. 


426  BACTERIOLOGY. 

The  results  obtained  in  the  health  department  labor- 
atories, as  well  as  elsewhere,  have  shown  that  in  a 
certain  proportion  of  cases  not  typhoid  fever  there 
occurs  a  delayed  moderate  reaction  in  a  1  to  10  dilution 
of  serum  or  blood  (the  proportion  originally  proposed 
by  Widal);  but  very  rarely,  if  ever,  excepting  in 
typhoid  fever,  or  at  least  typhoid  infection,  does  a 
complete  reaction  occur  in  this  dilution  within  jive 
minutes.  When  dried  blood  is  used  the  slight  ten- 
dency of  non-typhoid  blood  in  1  to  10  dilution  to 
produce  agglutination  is  increased  by  the  presence  of 
the  fibrinous  clumps,  and  perhaps  by  other  substances 
derived  from  the  disintegrated  blood-cells.  From  many 
cases  examined  by  Fraenkel,  Stern,  Forster,  Scholtz, 
ourselves  and  others,  it  has  been  found  that  in  dilutions 
of  1  to  20  or  more  a  decided,  quick  reaction  is  never 
produced  in  any  febrile  disease  other  than  that  due  to 
typhoid  infection,  while  in  typhoid  fever  such  a  distinct 
reaction  often  occurs  with  dilutions  of  1  to  50. 

The  mode  of  procedure,  therefore,  as  now  employed 
is  as  follows  :  The  test  is  first  made  with  the  typhoid 
bacillus  in  a  10  per  cent,  solution  of  serum  or  blood. 
In  the  case  of  serum,  one  part  of  serum  is  added  to 
nine  of  the  bouillon  culture.  With  dried  blood,  a  solu- 
tion of  the  blood  is  first  made,  and  the  final  dilution 
guessed  from  the  color  of  the  mixed  culture  and  blood 
solution.  To  obtain  an  idea  of  the  dilution  by  the 
color,  known  amounts  of  blood  are  dried  and  then 
mixed  with  definite  amounts  of  water  ;  the  colors  re- 
sulting are  fixed  in  the  memory  as  guides  for  future 
tests.  If  there  is  no  reaction — that  is  to  say,  if 
within  five  minutes  no  marked  change  is  noted  in  the 
motility  of  the  bacilli,  and  no  considerable  clumping 


BACILLUS  TYPHOSUS.  427 

occurs — nothing  more  is  needed;  the  result  is  negative 
as  far  as  this  specimen  is  concerned.  If  marked 
clumping  and  immobilization  of  the  bacilli  immedi- 
ately begin  and  become  complete  within  five  minutes, 
this  is  denominated  a  marked  immediate  typhoid  re- 
action, and  no  further  test  is  considered  necessary, 
though  it  is  always  advisable  to  confirm  the  reaction 
with  higher  dilutions  up  to  1  to  20  and  1  to  50.  If, 
however,  upon  examination  of  the  mixture  there  is  no 
marked  immediate  reaction,  but  the  bacilli  only  show 
in  the  first  few  minutes  an  inhibition  in  their  motility 
and  a  tendency  to  clump,  which  becomes  more  marked 
but  not  complete  within  five  minutes,  this  must  be 
tested  with  the  higher  dilution  of  1  to  20,  so  as  to 
measure  the  exact  strength  of  the  reaction.  If  in  the 
1  to  20  dilution  a  complete  reaction  takes  place  within 
thirty  minutes,  the  blood  is  considered  to  have  come 
from  a  case  of  typhoid  infection,  while  if  a  less  com- 
plete reaction  occurs  it  is  considered  that  a  probability 
only  of  typhoid  infection  has  been  established.  The 
time  allowed  for  the  development  of  the  reaction  with 
the  high  dilutions  is  by  many  from  one  to  two  hours, 
but  to  us  thirty  minutes  seem  safer.  Positive  results 
obtained  in  this  way  may  be  taken  to  be  conclusive 
unless  there  be  grounds  for  suspecting  that  the  reaction 
may  be  owing  to  a  previous  fairly  recent  attack.  The 
absence  of  reaction  in  one  examination  is  considered 
by  us  to  in  no  way  exclude  typhoid  infection.  If  the 
case  remains  clinically  doubtful,  the  examination  should 
be  repeated  within  a  few  days. 

Proportion  of  Oases  of  Typhoid  Fever  in  which  a  Defi- 
nite Reaction  Occurs  and  the  Time  of  its  Appearance. 
As  the  result  of  a  large  number  of  cases  examined  in 


428  BACTERIOLOGY. 

the  health  department  laboratories,  it  has  been  found 
that  about  20  per  cent,  gave  positive  results  in  the  first 
week,  about  60  per  cent,  in  the  second  week,  about  80 
per  cent,  in  the  third  week,  about  90  per  cent,  in  the 
fourth  week,  and  about  75  per  cent,  in  the  second 
month  of  the  disease.  In  88  per  cent,  of  the  cases  in 
which  repeated  examinations  were  made  (hospital  cases) 
a  definite  typhoid  reaction  was  present  at  some  time 
during  the  illness. 

Persistence  of  the  Reaction.  This  peculiar  property 
of  the  blood-serum  may  persist  in  persons  who  have 
recovered  from  typhoid  fever  for  a  number  of  months. 
Thus  a  definite  typhoid  reaction  has  been  reported  in 
serum  from  three  months  to  a  year,  and  a  slight  reac- 
tion, though  much  less  than  sufficient  to  establish  a 
diagnosis  of  typhoid  infection,  from  one  to  fifteen 
years,  after  convalescence  from  the  disease. 

The  Reaction  with  the  Blood-serum  of  Healthy  Persons 
and  of  Those  III  with  Diseases  other  than  Typhoid  Fever. 
An  immediate  marked  reaction  has  not  been  observed 
in  a  1  to  10  dilution  of  the  blood-serum  of  over  one 
hundred  healthy  persons  examined  in  the  health  depart- 
ment laboratories.  In  several  hundred  cases  of  diseases, 
not  believed  by  the  physicians  in  charge  to  be  at  the 
end  of  the  disease  typhoid  fever,  only  very  rarely  did 
the  serum  give  a  marked  immediate  reaction  in  a  1  to 
10  dilution,  and  here  in  the  light  of  past  experience,  I 
believe  a  typhoid  infection,  though  not  a  typical  typhoid 
fever,  to  have  existed.  These  results  have  been  con- 
firmed by  others,  the  question  of  dilution  having  been 
recently  made  the  subject  of  elaborate  investigations, 
with  the  view  of  determining,  if  possible,  at  what 
dilution  the  typhoid  serum  would  react  and  others 


BACILLUS  TYPHOSUS.  429 

would  not.  Thus,  Schultz  has  reported  lately  that 
among  100  cases  of  non-typhoid  febrile  diseases 
apparently  positive  results  were  obtained  in  19  with 
dilutions  of  1  to  5,  in  11  of  these  with  1  to  10,  in 
7  with  1  to  15,  in  3  with  1  to  20,  and  in  1  a  very  faint 
reaction  with  1  to  25;  whereas  in  as  many  cases  of  true 
typhoid  he  never  failed  with  dilutions  of  1  to  50.  In 
these  experiments  it  must  be  noted,  however,  that  the 
time-limit  was  from  one  to  two  hours.  A  faint  re- 
action with  a  1  to  25  dilution  with  a  time-limit  of  two 
hours  indicates  less  agglutinating  substance  than  an 
immediate  complete  reaction  with  a  1  to  10  dilution. 

From  an  experience  with  the  practical  application 
of  the  serum  test  for  the  diagnosis  of  typhoid  fever 
extending  over  three  years,  it  may  be  said  that  this 
method  of  diagnosis  is  simple  and  easy  of  perform- 
ance in  the  laboratory  by  an  expert  bacteriologist,  but 
it  is  not  to  be  recommended  for  routine  employment 
by  practising  physicians  as  a  clinical  test  unless  they 
have  had  experience;  that  with  the  modifications  as 
now  employed,  and  due  regard  to  the  avoidance  of  all 
possible  sources  of  error,  it  is  as  reliable  a  method  as 
any  other  bacteriological  test  at  present  in  use;  and 
that  as  such,  though  not  absolutely  infallible,  the  Widal 
test  is  an  indispensable  aid  to  the  clinical  diagnosis  of 
irregular  or  slightly  marked  typhoid  fever. 

The  Isolation  of  Typhoid  Bacilli  from  Suspected  Feces, 
Urine,  Blood,  Water,  etc.  In  the  bacteriological  study 
of  typhoid  infection  for  diagnostic  and  other  purposes, 
attempts  have  been  made  to  isolate  the  specific  bacilli 
from  the  blood,  rose-spots,  sweat,  urine,  feces,  and  by 
spleen  puncture.  Although  the  results  obtained  by 
puncture  of  the  spleen  have  been  encouraging  and  have 


430  BACTERIOLOGY. 

thrown  light  upon  the  distribution  of  the  organism  in 
the  body  daring  life,  yet  as  a  regular  means  of  diag- 
nosis it  is  to  be  discouraged,  on  account  of  the  possible 
danger  to  the  patient.  The  results  of  the  examination 
of  the  blood  and  rose-spots  of  typhoid  patients  have 
in  the  main  proved  unsatisfactory,  though  the  investi- 
gations of  some  of  the  later  observers  have  given  a 
number  of  positive  results  from  the  blood.  The  exam- 
ination of  the  urine  and  feces  of  typhoid  patients  has 
more  often  given  positive  results  than  the  blood, 
and  these  positive  results  have  become  more  fre- 
quent and  satisfactory  as  the  methods  for  differentiating 
the  bacillus  typhosus  have  grown  more  exact  and  re- 
fined. 

There  are  at  present  several  recently  devised  media 
employed  for  the  isolation  and  identification  of  the 
typhoid  bacillus,  which  are  much  better  than  any  of 
those  formerly  used.  These  are  the  Hiss,  Capaldi, 
and  Eisner  media.  In  the  hands  of  trained  bacteri- 
ologists they  give  satisfactory  results. 

THE  Hiss  MEDIA  : l  T/ieir  Composition  and  Prepa- 
ration. Two  are  used:  one  for  the  isolation  of  the 
typhoid  bacillus  by  plate  culture,  and  one  for  the 
differentiation  of  the  typhoid  bacillus  from  all  other 
forms  in  pure  culture  in  tubes. 

The  plating  medium  is  composed  of  10  grammes  of 
agar,  25  grammes  of  gelatin,  5  grammes  of  sodium 
chloride,  5  grammes  of  Liebig's  beef  extract,  10 
grammes  of  glucose,  and  1000  c.c.  of  water.  When 
the  agar  is  thoroughly  melted  the  gelatin  is  added  and 

1  This  description  is  taken  Irom  an  article  by  Dr.  Philip  Hanson  Hiss,  Jr., 
"  On  a  Method  of  Isolating  and  Identifying  Bacillus  Typhosus  and  Members 
of  the  Colon  Group  in  Semi-solid  Culture  Media,"  published  in  the  Journal 
of  Experimental  Medicine,  1897,  vol.  ii.  No.  6. 


BACILLUS  TYPHOSUS.  431 

completely  dissolved  by  a  few  minutes'  boiling.  The 
medium  is  then  titrated,  to  determine  its  reaction, 
phenol phthalein  being  used  as  the  indicator.  The 
requisite  amount  of  normal  hydrochloric  acid  or  sodium 
hydrate  solution  is  added  to  bring  it  to  the  desired 
reaction — i.  e.,  a  reaction  indicating  2  per  cent,  of  nor- 
mal acid.  To  clear  the  medium  add  one  or  two  eggs, 
well  beaten  it)  25  c.c.  of  water,  boil  for  forty-five  min- 
utes, and  filter  through  a  thin  filter  of  absorbent  cotton. 
Add  the  glucose  after  clearing.  The  reaction  of  the 
medium  is  most  important;  it  should  never  contain  less 
than  2  per  cent,  of  normal  acid. 

The  tube  medium  contains  agar,  5  grammes;  gelatin, 
80  grammes;  sodium  chloride,   5  grammes;  meat  ex- 

FIG.  54. 


Hiss'  plate  media:  Small  light  colony  (t)  is  composed  of  typhoid  bacilli; 
large  colony  (c)  of  colon  bacilli.    (From  Hiss.) 

tract,  5  grammes,  and  glucose,  10  grammes  to  the  litre 
of  water,  and  reacts  to  1.5  per  cent,  acid  by  the  indi- 
cator. The  mode  of  preparation  is  the  same  as  for  the 
plate  medium,  care  being  taken  always  to  add  the 


432  BACTE&IOLOGY. 

gelatin  after  the  agar  is  thoroughly  melted,  so  as  not 
to  alter  this  ingredient  by  prolonged  exposure  to  high 
temperature.  The  glucose  is  added  after  clearing. 
The  medium  must  contain  1.5  per  cent,  of  normal 
acid. 

Growth  of  the  Colonies.  The  growth  of  the  typhoid 
bacilli  in  plates  made  from  the  medium  as  above  de- 
scribed gives  rise  to  small  colonies  with  irregular  out- 
growth and  fringing  threads  (Figs.  54  and  55).  The 

FIG.  55. 


Colony  of  typhoid  bacilli  more  highly  magnified.    (Hiss.) 

colon  colonies,  on  the  other  hand,  are  much  larger, 
and,  as  a  rule,  are  darker  in  color  and  do  not  form 
threads.  The  growth  of  the  typhoid  bacilli  in  tubes 
produces  uniform  clouding  at  37°  C.  within  eighteen 
hours.  The  colon  cultures  do  not  give  the  uniform 
clouding,  and  present  several  appearances,  probably 
dependent  upon  differences  in  the  degree  of  their 
motility  and  gas-producing  properties  in  media.  Some 
of  the  varieties  of  the  colon  bacillus  grow  only  locally 


BACILLUS  TTPHOSUS.  433 

where  they  were  inoculated  by  the  platinum  needle. 
Others  grow  diffusely  through  the  medium,  but  owing 
to  the  production  of  gas  and  the  passage  of  gas-bubbles 
through  the  medium,  clear  streaks-  ramify  through  the 
otherwise  diffusely  cloudy  tube  contents.  This  charac- 
teristic appearance  is  not  produced  when  the  medium 
is  incorrect  in  reaction  or  in  consistency.  With  un- 
tried media  it  is  always  well  to  insert  a  platinum  wire 
into  the  tube  contents  and  stir  it  about ;  if  any  gas  is 
liberated  the  culture  is  not  one  of  the  typhoid  bacillus 
and  the  medium  is  not  correct. 

Method  of  Making  the  Test.  The  usual  method  of 
making  the  test  is  to  take  enough  of  the  specimen  of 
feces  or  urine — i.  e.,  from  one  to  several,  loops — and 
transfer  it  to  a  tube  containing  broth.  From  this 
emulsion  in  broth  five  or  six  plates  are  generally 
made  by  transferring  one  to  five  loops  of  the  emulsion 
to  tubes  containing  the  melted  plate  medium  and  then 
pouring  the  contents  of  these  tubes  into  Petri  dishes. 
These  dishes  are  placed  in  the  incubator  at  37°  C. 
and  allowed  to  remain  for  eighteen  to  twenty-four 
hours,  when  they  may  be  examined.  If  typical 
thread-forming  colonies  are  found  the  tube  medium 
is  inoculated  from  them  and  the  growth  in  the  tubes 
allowed  to  develop  for  about  eighteen  hours  at  37°  C. 
If  these  tubes  then  present  the  characteristic  clouding, 
experience  indicates  that  the  diagnosis  of  typhoid  may 
be  safely  made,  for  the  typhoid  bacillus  alone,  of  all 
the  organisms  investigated,  has  displayed  the  power 
of  giving  rise  both  to  the  thread- forming  colonies  in 
the  plating  medium  and  the  uniform  clouding  in  the 
tube  medium  when  exposed  to  a  temperature  of  37°  C. 
The  organisms  isolated  in  this  manner  have  been  sub- 

28 


434  B  A  OTERIO  LOGY. 

jectecT  to  the  usual  tests  for  the  recognition  of  the 
bacillus  typhosus,  and  have  always  corresponded  in 
all  their  reactions  to  those  given  by  the  typical  typhoid 
bacillus. 

ELSNER'S  METHOD.1  As  Eisner  himself  gives  no 
very  definite  details  as  to  the  steps  to  be  taken  in 
making  up  his  medium,  those  working  with  it  have 
developed  their  own  modifications.  We  found  the 
following  method  to  give  satisfactory  results : 

1.  Grate  0.5  kilogramme  of  small  potatoes  to  a  fine 
pulp,  add  1  litre  of  water,  and  allow  the  mixture  to 
stand  in  a  cool  place  over  night. 

2.  Mash  thoroughly  (meat-press  is  best)  and  strain 
through  a  fine  cloth.     This  must  be  done  when  the 
mixture  is  cold  or  the  swelling  of  the  starch-granules 
will  prevent  the  filtering  process. 

3.  Boil  the  filtrate  and  filter  again. 

4.  Add  10  per  cent,    of  gelatine  and  dissolve  by 
boiling. 

5.  Test  for  the  acidity.     Eisner  used  litmus  as  an 
indicator,  and  advised  that  the  medium  be  of  such  an 
acidity  as  to  require  the  addition  of  2.5  c.c.  of  deci- 
normal    hydroxide    solution    to    make   it  neutral.     If 
more  than  2.5  c.c.  are  required  the  acidity  must  be 
reduced   by    normal   sodic   hydrate   solution.      Abbot 
advises   using   phenolphtalein    as   the   indicator,    and 
making  the  reaction  such  that  3  c.c.  of  the  decinormal 
solution  will  neutralize  10  c.c.  of  the  medium. 

6.  Boil  and  clarify  with  an  egg. 

7.  Filter   first    through    cotton    and   then   through 
paper. 

i  Zeitschrift  fur  Hyg.  und  Infek.,  1896,  Bd.  xxi.  S.  25. 


BACILLUS  TYPHOSUS.  435 

8.  To   the    filtrate   add    1  per   cent,    of   potassium 
iodide.     (Use  a  solution  so  made  that  1  c.c.  is  equiva- 
lent to  1  gramme  of  the  salt.) 

9.  Decant  into  tubes  and  sterilize. 

One  of  the  most  important  points  in  working  with 
this  medium  is  that  the  incubator  must  be  kept  at  a  con- 
staut  temperature  of  from  22°  to  24°  C.  If  the  plates 
be  put  away  before  the  gelatin  has  thoroughly  cooled, 
or  if  the  room  becomes  a  very  little  too  warm  at  any 
time  during  the  colony  growth,  so  as  to  soften  the  gel- 
atin, both  the  typhoid  and  the  colon  bacilli  will  develop 
threads  or  become  oval,  and  thus  the  characteristic  dif- 
ferentiation will  be  lost.  Care  must  be  taken,  also, 
that  the  room  in  which  the  plates  are  examined  be  not 
too  warm.  This  causes  great  inconvenience  during  the 
summer  months  in  most  parts  of  the  United  States, 
and  requires  special  methods  for  keeping  a  room  at  the 
proper  temperature  and  keeping  the  plates  cool  during 
their  examination. 

The  iodide  of  potash  prevents  the  great  majority  of 
bacteria,  especially  the  liquefiers,  from  developing;  in 
fact,  little  but  colon  and  typhoid  bacilli  appear  on  the 
plates.  This  is  one  of  the  chief  advantages  of  the 
medium  in  the  examination  of  both  water  and  feces. 

Appearance  of  the  Colonies.  The  colon  is  the  first  to 
develop;  the  colonies  are  rough  and  granular  in  appear- 
ance and  greenish-brown  in  color;  for  the  greater  part 
the  colonies  are  on  or  near  the  surface.  The  typhoid 
develop  later,  and  their  colonies  usually  show  the  clas- 
sical "  dew-drop  "  appearance — small,  white,  gleaming, 
generally  without  variation  in  substance,  but  occasion- 
ally slightly  granular.  This  point  causes  some  trouble 
to  one  first  using  the  medium,  as  the  young  colon  colo- 


436  BACTERIOLOGY. 

nies  sometimes  present  much  the  same  appearance. 
However,  it  was  found  that  the  typhoid  colonies  usu- 
ally grew  near  the  surface,  while  the  colon  colonies, 
which  are  small  and  white,  are  almost  invariably  very 
deep.  This  is  especially  true  after  forty-eight  hours; 
therefore,  if  one  is  not  sure  of  a  colony  from  the 
appearance,  an  attempt  to  "fish"  it  will  usually 
identify  it. 

To  one  familiar  with  the  medium,  the  characteristic 
appearance  of  a  plate  when  typhoid  is  present  is  almost 
unmistakable,  and  it  would  seem  that  one  would  be 
almost  sure  to  find  the  typhoid  in  some  one  of  a 
series  of  plates,  even  if  there  were  but  few  in  the 
specimen. 

Eisner  says  that  the  typhoids  do  not  develop  for 
forty-eight  hours.  Although  the  differentiation  is 
more  accurate  after  that  time,  still  for  practical 
methods  of  work  twenty-four  hours  was  found  to  be 
quite  sufficient.  When  the  plates  are  first  taken  from 
the  incubator  the  diagnosis  is  not  quite  as  certain,  for 
the  colon  colonies  will  not  have  developed  the  charac- 
teristic color;  but  if  the  plates  be  allowed  to  stand  in 
the  light  for  a  couple  of  hours  the  diagnosis  will  be 
found  quite  easy.  After  forty-eight  hours  all  the  large 
colonies  will  be  colon  and  most  of  the  small  ones 
typhoid,  if  there  be  any  typhoid  present.  Even  after 
several  days'  standing  in  the  ice-chest  the  typhoid  colo- 
nies do  not  develop  color. 

Except  for  the  difficulty  in  obtaining  an  exact  tem- 
perature for  growth  and  a  cool  room  for  examination, 
this  method  was  found  very  satisfactory. 

THE  CAPALDI  PLATE  MEDIUM.  In  his  original 
paper,  Capaldi  gives  the  following  recipe: 


BACILLUS  TYPHOSUS.  437 

Aqua  dest t          .  1000 

Gelatin 20 

Mannite  (grape-sugar)      .         .         .         .         .  10 

Sodium  chloride        ......  5 

Potassium  chloride  ......  5 

Boil,  filter,  add  2  per  cent,  agar  and  10  c.c.  of  normal  sodic 
hydrate  solution  ;  boil,  filter,  and  sterilize. 

In  making  up  the  medium  for  work  the  only  varia- 
tion was  that  the  agar  was  added  when  the  gelatin  was 
put  in  in  the  original  recipe,  and  the  gelatin  was 
added  after  the  first  filtration. 

The  Capaldi  medium  is  usually  employed  for  surface 
cultures,  but  can  be  inoculated  while  melted  in  the 
tubes.  Plates  may  be  made  beforehand,  so  that  they 
are  ready  for  use  when  the  specimen  comes  in.  As 
these  plates  are  to  be  kept  at  37°  C.,  the  difficulties  in 
regard  to  temperature  are  avoided;  but,  unlike  the 
Eisner  plates,  other  organisms  beside  the  colon  and 
typhoid  develop  and  may  cause  some  confusion.  In 
making  the  plates  one  or  two  are  inoculated  by  gently 
carrying  across  their  surface  a  platinum  loop  of  feces 
or  urine.  Others  are  then  inoculated  with  a  loop  of 
urine  or  much  diluted  feces.  In  this  way  we  are  apt 
to  have  some  plates  with  just  the  right  amount  of 
colonies. 

Appearance  of  the  Colonies.  Capaldi  thus  describes 
the  differentiation:  Typhoid:  Small,  gleaming,  transpa- 
rent, almost  colorless  colonies  (by  reflected  light,  blue). 
Colon:  Large,  milky  colonies  (reflected  light,  brown). 

In  using  the  medium  it  was  found  that  even  in  a 
pure  plate  of  typhoid  the  colonies  vary  much  in  size 
and  appearance,  while  different  typhoids  show  indi- 
vidual differences  in  growth.  In  general,  a  medium- 
sized,  gray-white  colony,  with  a  few  refractive  granules, 


438  BACTERIOLOGY. 

is  the  typhoid  (Fig.  56).  However,  it  is  often  trans- 
parent, without  the  refractive  granules;  sometimes 
with  a  nuclear  centre,  and  sometimes  of  equal  con- 
sistency throughout.  Streptococci  simulate  typhoid, 
but  a  high-power  lens  will  show  the  coccus. 

FIG.  56. 


Colonies  of  typhoid  bacilli,  on  Capaldi  medium,  slightly  magnified. 

Colon  colonies  are  usually  much  larger  than  the 
typhoid;  a  decided  brown  color,  very  large,  refractive 
granules,  and  in  general  quite  different  in  appearance 
(Fig.  57). 

The  best  way  to  work  with  the  Capaldi  medium  is  to 
make  several  plates  with  different  typhoid  cultures, 
observe  carefully  all  the  variations  in  the  colonies,  and 
bear  these  in  mind  when  working  with  the  mixed 
plates.  After  these  precautions  have  been  taken  the 


BACILLUS  TYPHOSUS. 


439 


medium  will  be  found  very  satisfactory.  The  colonies, 
as  a  rule,  appear  characteristically  in  twelve  to  eigh- 
teen hours,  and  thus  give  a  quick  method  of  diagnosis. 
The  two  media  together  (Capaldi  and  Eisner)  work 
excellently,  as  one  is  an  aid  to  the  other.  When 
many  colonies  of  the  typhoid  bacilli  were  present  the 

FIG.  57. 


Colonies  of  colon  bacilli.    Capaldi  medium  slightly  magnified. 

differentiation  was  usually  easily  seen  upon  both  media, 
and  the  two  together  made  diagnosis  almost  certain. 
The  bacilli  from  the  suspected  typhoid  colonies  can  be 
quickly  tested  sufficiently  for  practical  purposes  on  the 
Hiss  tube  medium  and  by  the  reaction  between  the 
bacilli  and  the  serum  from  an  immunized  horse. 

As  to  the  comparative  merits  of  these  three  media, 
it  is  probably  safe  to  say  that  any  one  of  them  will,  in 
the  hands  of  one  accustomed  to  them,  reveal  the  typhoid 
bacilli,  if  they  are  present,  except  perhaps  when  they 


440  -B4  CTERIOL  OGY. 

exist  in  only  the  most  minute  numbers.  The  Eisner 
method  has  the  objection  that  it  is  very  difficult  to  work 
with  in  hot  weather.  The  Hiss  plate  medium  has  the 
objection  that  it  is  a  difficult  medium  to  prepare.  If 
the  acidity  is  not  just  right  the  thread  outgrowths  do 
not  appear.  Indeed,  the  only  sure  way  is  to  test  a  new 
batch  of  medium  with  a  pure  culture  and  alter  the 
reaction  until  the  culture  grows  correctly.  A  very  few 
varieties  of  the  typhoid  bacillus  do  not  produce  typical 
thread  outgrowths  from  the  colonies. 

The  Capaldi  medium  has  the  objection  that  some  of 
the  typhoid  and  some  of  the  colon  colonies  frequently 
look  much  alike.  If  one,  however,  will  always  pick 
out  the  colonies  which  look  most  like  the  typhoid,  it 
will  usually  turn  out  that  typhoid  bacilli  have  been 
obtained  if  any  were  present.  Personally,  for  general 
use  I  prefer  the  Capaldi  medium  for  the  plate  cultures 
and  the  Hiss  tube  medium  for  identifying  the  bacilli 
obtained.  Through  these  media  we  are  now  in  a  posi- 
tion to  obtain  and  identify  typhoid  bacilli  from  feces, 
urine,  etc.,  within  forty-eight  hours. 

Recently  numerous  investigations  have  been  carried 
out  to  discover  how  frequently  and  at  what  period  in 
typhoid  fever  cases  bacilli  were  present  in  the  feces 
and  urine.  In  the  laboratory  Hiss  has  recently  exam- 
ined the  feces  in  forty- three  consecutive  cases,  thirty- 
seven  of  which  were  in  the  febrile  stage  and  six  con- 
valescent. In  a  number  of  instances  only  one  stool  was 
examined,  but  even  under  these  adverse  conditions  the 
average  of  positive  results  in  the  febrile  stage  was  66.6 
per  cent. 

Out  of  26  cases  of  typhoid  fever  in  hospitals  exam- 
ined;  21  were  in  the  febrile  stage  and  5  convalescent. 


BACILLUS  TYPHOSUS.  441 

In  the  febrile  cases  in  17  the  presence  of  typhoid  bacilli, 
often  in  great  numbers,  was  demonstrated.  Thus  in 
these  carefully  followed  cases  the  statistics  show  over 
80  per  cent,  of  the  febrile  cases  positive.  The  bacilli 
were  isolated  from  these  cases  as  early  as  the  sixth  day, 
and  as  late  as  the  thirtieth  day,  and  in  a  case  of  relapse 
on  the  forty-seventh  day  of  the  disease.  The  conval- 
escent cases  gave  uniformly  negative  results,  the  earliest 
examination  having  been  made  on  the  third  day  after 
the  disappearance  of  the  fever.  The  bacilli  seemed  to 
be  more  numerous  in  the  stools  from  about  the  tenth  or 
twelfth  day  on.  These  observations,  with  regard  to  the 
appearance  of  the  bacilli  in  the  stools  during  the  febrile 
stage  and  their  usually  quick  disappearance  after  the 
defervescence,  have  been  confirmed  by  others.  In 
several  cases  in  which  no  Widal  reaction  was  demon- 
strated the  bacilli  were  isolated.  From  private  sources 
between  the  seventh  and  twenty-first  day  of  the  disease, 
experience  thus  far  obtained  seems  to  indicate  that  the 
bacilli  may  be  obtained  from  about  25  per  cent,  of  all 
cases  on  the  first  examination  and  from  about  75  per 
cent,  after  repeated  examinations.  In  some  samples  of 
feces  typhoid  bacilli  die  out  within  twenty -four  hours; 
in  others  they  remain  alive  for  days  or  even  weeks. 
This  seems  to  depend  on  the  bacteria  present  in  the 
feces  and  upon  its  chemical  formation.  Probably  the 
presence  of  typhoid  bacilli  in  some  stools  and  their  ab- 
sence in  others  must  be  explained  largely  upon  the 
characteristics  of  the  intestinal  contents.  The  short 
life  of  the  typhoid  bacillus  in  many  specimens  of  feces 
suggests  that  stools  be  examined  as  quickly  as  possible. 
In  fact,  unless  the  physician  wishes  to  take  the 
trouble  to  have  the  sample  of  feces  sent  immediately 


442  BACTERIOLOGY. 

to  the  laboratory,  it  is  hardly  worth  while  for  the  bac- 
teriologist to  take  the  trouble  to  make  the  test. 

Typhoid  Bacilli  in  the  Urine.  Of  even  more  interest 
than  the  presence  of  the  bacilli  in  feces  is  their  frequent 
occurrence  in  great  numbers  in  the  urine.  The  results 
of  the  examinations  of  others  as  well  as  those  of  our 
own  indicate  that  the  typhoid  bacilli  are  not  apt  to  be 
found  in  the  urine  until  the  beginning  of  the  third  week 
of  the  fever,  and  may  not  appear  until  much  later. 
From  this  on  to  convalescence  they  appear  in  about  25 
per  cent,  of  the  cases,  and  usually  in  pure  culture  and  in 
enormous  numbers.  Of  nine  positive  cases  examined 
by  Richardson1  two  died  and  seven  were  discharged. 
At  the  time  of  their  discharge  their  urine  was  loaded 
with  typhoid  bacilli.  We  have  noted  similar  cases.  In 
one  the  bacilli  persisted  for  five  weeks.  Undoubtedly 
in  some  cases  they  persist  for  months.  When  we  think 
of  the  chances  such  cases  have  to  spread  infection  as 
they  pass  from  place  to  place,  we  begin  to  realize 
how  epidemics  can  start  without  apparent  cause.  The 
more  we  investigate  the  persistence  of  bacteria  in  con- 
valescent cases  of  disease  the  more  difficult  the  preven- 
tion of  their  dissemination  is  seen  to  be.  The  disin- 
fection of  the  urine  should  always  be  looked  after  in 
typhoid  fever,  and  convalescents  should  not  be  allowed 
to  go  to  places  where  contamination  of  the  water-supply 
is  possible  without  at  least  warning  them  of  the  neces- 
sity of  great  care  in  disinfecting  their  urine  and  feces 
for  some  weeks.  Richardson  made  the  interesting  dis- 
covery that  after  washing  out  the  bladder  with  a  very 
weak  solution  of  bichloride  of  mercury  the  typhoid 
bacilli  no  longer  appeared  in  the  urine. 

1  Journal  of  Experimental  Medicine,  May,  1898. 


BACILLUS  TYPHOSUS.  443 

The  Detection  of  Typhoid  Bacilli  in  Water.  This  sub- 
ject is  considered  on  pages  247  and  248.  There  is 
absolutely  no  doubt  that  the  contamination  of  streams 
and  reservoirs  is  the  frequent  cause  of  the  outbreak  of 
epidemics  of  typhoid  fever,  but  the  actual  finding  and 
isolation  of  the  bacilli  is  a  very  rare  occurrence.  This 
is  owing  to  the  contamination  often  having  occurred 
and  passed  away  before  the  bacteriological  examination 
is  undertaken,  and  also  because  of  the  great  difficulties 
met  with  in  detecting  a  few  typhoid  bacilli  when  they 
are  associated  with  large  numbers  of  other  bacteria. 
The  greater  the  amount  of  contamination  which  is 
thrown  into  the  water,  and  the  shorter  the  time  which 
elapses  between  the  infection  and  the  drinking  of  the 
water,  the  greater  is  the  danger. 

The  typhoid  bacillus  and  the  colon  bacillus  of 
Escherich  resemble  each  other  in  many  respects.  The 
characteristics  of  each,  which  allow  us  to  differentiate 
the  one  from  the  other,  will  be  considered  at  the  end  of 
the  description  of  the  colon  bacillus. 


CHAPTER   XXIV. 

BACILLUS   COLI   COMMTJNIS   (OR   COLON   BACILLUS   OF 
ESCHERICIl). 

THIS  organism  was  first  described  by  Emmerich 
(1885),  who  obtained  it  from  the  blood,  various  organs, 
and  intestinal  discharges  of  cholera  patients  at  Naples. 
It  was  afterward  found  by  Escherich  (1886)  in  the 
feces  of  healthy  milk-fed  infants  and  by  Weisser  in 
the  alvine  discharges  of  healthy  men.  It  has  since 
been  demonstrated  to  be  a  normal  inhabitant  of  the 
intestines  of  man  and  of  many  of  the  lower  animals. 

Morphology.  The  size  and  shape  of  the  bacillus  coli 
varies  considerably  in  its  morphology  according  to  the 
sources  and  the  culture  media  from  which  it  is  obtained. 
The  typical  form  is  that  of  short  rods  with  rounded 
ends,  from  0.4/z  to  0.7/*  in  diameter  by  I/JL  to  3//  in 
length;  but  sometimes  the  rods  are  so  short  as  to  be 
almost  spherical,  resembling  micrococci  in  appearance, 
and,  again,  they  are  somewhat  oval  in  form  or  are  seen 
as  threads  of  6^  or  more  in  length.  The  various 
forms  may  often  be  associated  in  the  same  culture  (Fig. 
58).  The  bacilli  may  occur  as  single  cells  or  as 
pairs  joined  end-to-end,  rarely  as  short  chains.  In 
unfavorable  culture  media  in  stained  preparations  they 
may  present  unstained  spaces  (vacuoles)  and  more  in- 
tensely stained  portions  at  the  extremities,  closely 
resembling  spores,  but  these  are  due,  according  to 


BACILLUS  COLI  COMMUNIS.  445 

Escherich,  to  degenerative  changes  in  the  protoplasm. 
The  colon  bacillus  does  not  form  spores.  There  is 
nothing  in  the  morphology  of  this  bacillus  which  is 
characteristic  or  which  may  aid  in  its  identification, 
for  in  this  respect  it  simulates  many  other  organisms. 

FIG.  58. 


Colon  bacilli.    Twenty-four-hour  agar  culture.    X  1100  diameters. 

The  colon  bacillus  stains  readily  with  the  ordinary 
aniline  colors;  it  is  quickly  decolorized  by  Gram's 
method. 

Biology.  It  is  an  aerobic,  facultative  anaerobic,  non- 
liquefying  bacillus.  It  is  motile,  but  its  movements 
are  so  sluggish  that  a  positive  opinion  is  often  difficult, 
being  exhibited  often  by  one  or  two  individuals,  in  fresh 
cultures,  and  at  a  high  temperature  only.  These  move- 
ments are  produced  by  flagella,  which  may  be  demon- 
strated by  Loffler's  method  of  staining,  though  not 
usually  in  such  numbers  as  are  seen  to  occur  on  the 
bacillus  typhosus. 


446  BACTERIOLOGY. 

Growth  on  Gelatin.  In  gelatin  plates  colonies  are 
developed  in  from  twenty-four  to  forty-eight  hours, 
which  vary  considerably  in  their  appearance  according 
to  their  age,  and  in  different  cultures  in  the  same 
medium.  They  resemble  greatly  the  colonies  of  the 
typhoid  bacillus,  except  that  they  are  somewhat  larger 
for  the  same  period  of  growth.  When  located  in  the 
depths  of  the  gelatin  and  examined  by  a  low-power 
lens  they  are  at  first  seen  to  be  finely  granular,  almost 
homogeneous,  in  structure  round,  and  of  a  pale  yellow- 
ish to  brownish  color;  later  they  become  larger,  denser, 
darker,  and  more  coarsely  granular.  In  shape  they 
may  be  round,  oval,  or  "  whetstone-like. "  The  super- 
ficial colonies  appear  as  small,  dry,  irregular,  flat, 
blue- white  points  that  are  commonly  somewhat  dentated 
at  the  margin. 

In  stab  cultures  on  gelatin  the  growth  usually  takes 
the  form  of  a  nail  with  a  flattened  head,  the  surface 
extension  generally  reaching  out  rapidly  to  the  sides 
of  the  tube. 

On  Nutrient  Agar  and  Blood-serum.  On  nutrient 
agar  and  blood-serum  an  abundant,  soft,  white  layer  is 
quickly  developed  in  the  incubator,  but  the  growth 
is  not  characteristic. 

In  Bouillon.  In  bouillon  the  bacillus  coli  produces 
diffuse  clouding  with  sedimentation;  in  some  cultures 
a  tendency  to  pellicle  formation  on  the  surface  is  seen 
occasionally.  In  old  cultures,  in  the  absence  of  sugar, 
the  reaction  becomes  alkaline,  and  a  decided  fecal  odor 
may  be  noticed. 

The  colon  bacillus  produces  indol  in  bouillon  and  in 
peptone  solutions,  this  reaction  being  most  pronounced 
after  a  week's  development  in  the  incubator.  It  pos- 


BACILLUS  COLI  COMMUNIS.  447 

sesses  also  a  considerable  reducing  power,  converting 
nitrates  into  nitrites,  as  may  be  demonstrated  by  the 
addition  of  sulphuric  acid  in  the  proper  proportion  to 
a  bouillon  or  peptone  culture,  when  a  pink  coloration 
results. 

On  Potato.  On  potato  the  growth  is  rapid  and 
abundant,  appearing  after  twenty-four  to  thirty-six 
hours  in  the  incubator  as  a  yellowish-brown  to  dark 
cream-colored  deposit  covering  the  greater  part  of  the 
surface.  But  there  are  considerable  variations  from 
the  typical  growth  on  potato;  there  may  be  no  growth 
at  all,  or  it  may  be  scanty  and  of  a  white  color.  These 
variations  are  due  at  times  to  the  bacillus,  but  more 
often  to  variations  in  the  potato. 

Gas-production.  The  bacillus  coli  grows  rapidly 
in  media  containing  glycerin  and  sugar,  particularly 
glucose,  causing  active  fermentation  with  liberation  of 
carbonic  acid  and  hydrogen  gas.  Cultivated  in  solid 
media,  to  which  glucose  has  been  added,  the  gas-pro- 
duction is  recognized  by  the  appearance  of  numerous 
bubbles  along  and  about  the  points  of  growth ;  in  fluid 
media  it  may  be  demonstrated  in  the  fermentation- tube. 
Grown  on  lactose-litmus-agar,  the  colonies  are  pink 
and  the  color  of  the  surrounding  medium  is  changed 
from  blue  to  red,  showing  the  production  of  acid. 

Milk  is  coagulated  by  the  growth  of  the  bacillus 
coli  after  twenty-four  to  seventy-three  hours  in  the  in- 
cubator, with  the  production  of  gas  and  acid;  very 
rarely  acid  may  be  produced  and  no  coagulation  occur. 
The  coagulation  of  the  milk  is  hastened  by  warming. 

The  thermal  death-point  of  the  colon  bacillus  from 
feces  was  found  by  Weisser  to  be  60°  C.,  the  time  of 
exposure  being  ten  minutes.  When  the  bacilli  from 


448  BACTERIOLOGY. 

a  bouillon  culture  were  dried  upon  thin  glass  covers 
they  failed  to  grow  after  twenty-four  hours  (Weisser). 
Waliczek  found  that  when  dried  upon  pieces  of  sterile 
filter-paper  they  failed  to  grow  at  the  end  of  eighteen 
hours.  These  results  give  confirmation  to  the  view 
that  the  colon  bacillus  does  not  form  spores. 

Pathogenesis.  The  colon  bacillus  is  pathogenic  in 
varying  degrees  for  test  animals,  though  the  results  of 
the  inoculations,  as  with  the  typhoid  bacillus,  cannot 
always  be  predicted  with  certainty.  Intraperitoneal 
injections  of  from  0.1  to  1  c.c.  of  fresh,  virulent  cul- 
tures usually  produce  death  in  mice  at  the  end  of  from 
one  to  eight  days,  but  death  does  not  invariably  follow. 
The  more  rapidly  death  ensues  the  greater  the  number 
of  bacilli  found  in  the  body;  they  are  always  more 
abundant  in  the  abdominal  cavity  than  in  the  blood; 
in  other  words,  the  result  is  to  be  attributed  to  the 
toxic  rather  than  to  the  infective  properties  of  the 
culture  used.  But  the  fact  that  the  bacilli  are  found 
in  the  blood  and  internal  organs  when  death  rapidly 
follows  inoculation  proves  that  they  do  multiply  to 
some  extent  in  the  body.  When  less  virulent  cultures, 
however,  are  injected  and  death  results,  this  is  due  to 
the  poisonous  products  formed  by  the  bacilli  and  given 
up  at  their  death.  The  lesions  produced  are  those  of 
enteritis  :  the  duodenum  and  jejunum  are  found  to  con- 
tain fluid,  the  spleen  is  somewhat  enlarged,  and  there 
is  marked  hypersemia  and  ecchymosis  of  the  small  in- 
testines, together  with  swelling  of  Peyer's  patches. 

Intraperitoneal  and  intravenous  inoculation  of  guinea- 
pigs  and  rabbits  may  also  produce  death,  which,  when 
it  follows,  usually  takes  place  within  the  first  forty-eight 
hours,  accompanied  by  a  decided  fall  of  temperature, 


BACILLUS  COLI  COMMUNIS.  449 

the  symptoms  of  enteritis,  diarrhoea,  etc.,  and  finally 
fibro-purulent  peritonitis. 

When  subcutaneous  inoculations  of  mice  and  guinea- 
pigs  are  made  it  requires  the  introduction  of  much 
larger  quantities  of  the  culture  to  produce  infection; 
in  rabbits  this  is  followed  only  by  abscess  formation  at 
the  point  of  inoculation.  Dogs  and  cats  are  similarly 
affected. 

Bazy  and  Guyon  have  succeeded  in  producing  infec- 
tion of  the  bladder  in  animals  by  injection  of  pure 
cultures  into  the  blood  with  simultaneous  tying  of  the 
ureters;  Albaran  and  Halle  have  caused  cystitis  and 
pyelonephritis  by  direct  injections  into  the  bladder  and 
ureters,  the  urine  being  artificially  suppressed;  Chassin 
and  Roger  produced  angiocholitis  and  abscess  of  the 
liver  in  the  same  way.  Loruelle,  Fraenkel,  and  Bar- 
hacci,  by  injuring  or  tying  the  intestines  and  intro- 
ducing dirt  into  the  abdominal  cavity,  with  or  without 
the  simultaneous  injection  of  cultures  of  the  colon 
bacillus,  succeeded  in  causing  diffuse  peritonitis  in 
animals.  Akermann  produced  osteomyelitis  in  young 
rabbits  by  intravenous  injections  of  cultures.  So  far 
all  attempts  to  produce  experimental  infection  of  the 
intestines  by  the  ingestion  of  cultures  of  the  colon 
bacillus  have  failed  to  give  positive  results  (Emmerich 
and  Korkunoff). 

Certain  peculiar  effects  have  been  observed  by  Black- 
stein  and  by  Gilbert  and  Lion  as  the  result  of  intra- 
venous inoculation  of  rabbits  with  pure  cultures  of  the 
bacillus  coli,  which  are  worthy  of  note.  The  former 
of  these  authors  found,  from  eight  to  thirty-eight  days 
after  injection,  that  the  liver  frequently  contained 
opaque,  whitish  or  yellowish-white  spots,  and  streaks 

29 


450  BACTERIOLOGY. 

of  irregular  size  and  shape,  giving  a  peculiar  mottling 
to  the  organ  when  present  in  large  numbers.  By  micro- 
scopical examination  these  were  found  to  represent 
places  where  the  liver  cells  had  undergone  necrosis, 
accompanied  by  emigration  of  leucocytes,  and  the  cells 
about  them  were  in  a  condition  of  fatty  degeneration. 
In  sections  of  the  liver,  masses  of  the  bacilli  were  dis- 
covered in  and  about  the  necrotic  foci.  The  bacilli 
were  not  found  generally  distributed  through  the  body, 
but  only  in  the  bile,  liver,  and  occasionally  in  the 
spleen.  Gilbert  and  Lion  found  in  addition  that  hemi- 
plegia  and  paraplegia  were  often  produced  in  conse- 
quence of  atrophy  of  the  cells  of  the  cord.  These 
observations  have  been  confirmed  by  Thoinot  and 
Massilin,  but  in  their  experiments  the  nerve-lesions 
were  not  commonly  present. 

From  experiments  on  animals  it  would,  therefore, 
appear  that  the  true  explanation  of  the  palhogensis  of 
the  colon  bacillus  is  undoubtedly  to  be  found  in  the 
toxic  effects  of  the  chemical  products  of  the  organism 
rather  than  in  its  mechanical  presence  in  the  tissues. 

Variation  in  Virulence.  The  virulence  of  the  colon 
bacillus  varies  considerably  as  derived  from  different 
sources.  An  attempt  has  been  made  to  establish 
certain  rules  for  this.  Thus,  Lesage  and  Macaigne 
express  the  opinion  that  when  obtained  from  a  healthy 
body  it  is  only  slightly  virulent,  while  that  isolated 
from  a  diseased  person  is  much  more  virulent.  The 
infective  power  is  thought  to  bear  a  definite  relation 
to  the  severity  of  the  disease  with  which  the  organism 
is  associated;  for  instance,  to  be  greatest  in  cultures 
taken  from  cholera  patients  and  least  in  those  obtained 
from  pus.  Dreyfus  also  confirms  this  view.  He  found 


BACILLUS  CO  LI  COMMUNIS.  451 

by  experiment  that  1  c.c.  of  a  fresh  bouillon  culture 
of  the  B.  coli  from  normal  feces  was  required  to  kill 
guinea-pigs  by  intraperitoneal  and  rabbits  by  intraven- 
ous injection,  whereas  less  than  one-fifth  as  much  of  a 
culture  from  a  fatal  case  of  cholera  nostras  was  suf- 
ficient to  kill  the  same  animal;  but  this  rule  has  prob- 
ably many  exceptions,  even  if  it  be  true  in  some  cases. 

All  observers,  however,  agree  that  the  virulence 
of  the  B.  coli  is  diminished  by  continued  cultivation 
through  successive  generations,  and  that  it  is  increased 
by  passage  through  animals. 

Immunization.  Immunization  against  colon  infec- 
tion is  comparatively  easy  to  produce  in  the  usual  way 
by  the  inoculation  of  gradually  increasing  doses  of 
cultures  of  the  living  bacilli  or  dead  bacilli. 

Occurrence  in  Man  and  Animals.  The  bacillus  coli 
communis  is  a  common  inhabitant  of  the  intestinal 
canal  in  man  and  in  many  animals.  According  to 
Fremlin,  it  is  found  normally  in  dogs,  mice,  and  rab- 
bits, but  not  in  rats,  pigeons,  or  guinea-pigs.  Accord- 
ing to  Dyas  and  Keith,  it  occurs  in  goats,  rabbits,  dogs, 
cats,  swine,  and  cows,  but  not  in  horses.  Grimbert 
claims  to  have  found  it  in  the  intestines  of  almost  all 
domestic  animals,  and  in  the  mouth  as  well  as  the  in- 
testines of  man.  It  is  also  frequently  found  in  water 
and  food  (milk,  etc.),  so  that  it  is  one  of  the  most  wide- 
spread saprophytic  bacteria  known.  Formerly  it  was 
thought  that  the  presence  of  the  B.  coli  in  water  was 
sufficient  proof  of  its  contamination  by  feces;  but  the 
recent  investigations  of  Weichselbaum,  Kruse,  Beck- 
mann,  and  Refith  would  seem  to  show  that  there  are 
no  grounds  for  this  assumption,  as  the  colon  bacillus 
may  reach  the  water  from  many  different  sources. 


452  BACTERIOLOGY. 

From  its  common  seat  in  the  intestines  it  may, 
under  favoring  conditions,  penetrate  other  organs  after 
death — which  fact  may  account  for  its  being  found 
so  often  at  autopsy  in  the  interior  of  the  body;  but  it 
may  also  be  absorbed  during  life,  more  especially  if 
there  is  obstruction  of  the  intestines  or  if  the  mucosa 
has  been  deprived  of  its  epithelium.  For  this  reason, 
no  doubt,  the  B.  coli  is  so  frequently  found  in  cholera, 
typhoid  fever,  and  dysentery,  producing  often  a  second 
infection.  The  absorption  of  the  colon  bacillus  from 
the  intestinal  canal  plays  an  important  part,  probably, 
in  the  production  of  many  diseases,  such  as  cystitis 
and  other  inflammatory  affections.  It  has  been  con- 
sidered to  be  the  cause  of  epidemic  infectious  enteritis 
and  cholera  nostras,  this  assumption  being  based  upon 
the  facts  that  the  colon  bacillus  in  these  diseases  is 
found  in  greater  abundance  than  usual  in  the  alvine 
discharges  and  often  in  pure  culture ;  that  it  then  pos- 
sesses an  increased  virulence,  and  that  it  often  pene- 
trates the  interior  organs,  as  has  been  shown  by  autop- 
sies. But  the  conclusion  drawn  from  these  facts  as  to 
the  etiology  of  the  diseases  above  mentioned  is  not 
positive,  though  it  cannot  be  denied  that  under  certain 
conditions  the  colon  bacillus  may  be  productive  of  dis- 
ease. This  is  brought  about,  according  to  the  com- 
monly accepted  view,  either  by  an  increase  of  virulence 
of  the  B.  coli  normally  present  in  the  intestines  or  by 
the  introduction  of  especially  virulent  bacilli  in  the 
food.  The  colon  bacillus  has  also  been  assumed  to  be 
the  cause  of  cholera  infautum;  but  the  investigations 
of  Booker,  Baginsky,  Escherich,  and  Fliigge  would 
seem,  to  indicate  that  this  disease  is  of  a  much  more 
complicated  origin.  The  B.  coli,  moreover,  is  associ- 


BACILLUS  COLI  COMMUNIS.  453 

ated  with  dysentery,  probably  as  a  secondary  affection, 
as  in  amoebic  or  tropical  dysentery.  It  is  also  found 
frequently  in  cases  of  diffuse  and  circumscribed  peri- 
tonitis, appendicitis,  etc.,  either  alone  or  together  with 
other  bacteria  which  play  a  part  in  the  etiology  of  these 
diseases  along  with  certain  chemical  ferments  and  toxins 
and  foreign  bodies  in  the  intestines.  The  origin  of  in- 
fections of  the  gall-ducts  (with  at  times  the  production 
of  gallstones)  and  multiple  abscess  of  the  liver  is  also 
explained  in  this  way  by  Dmochowski  and  Jauowski, 
though,  according  to  Letienne,  the  mere  presence  of 
the  B.  coli  in  the  bile,  in  which  it  has  been  found 
under  normal  conditions,  is  not  sufficient  to  account 
for  these  affections.  Puerperal  fever  is  not  infre- 
quently caused  by  the  colon  bacillus  by  infection  of 
the  vagina  or  uterus.  Other  diseases  to  which  the 
colon  bacillus  seems  to  stand  in  a  certain  relation, 
though  rarely,  are  :  Endocarditis,  meningitis,  tropical 
abscess  of  the  liver,  bronchopneumoriia  and  an  irregu- 
lar type  of  lobar  pneumonia,  fetid  bronchitis,  chronic 
amygdalitis,  and  abscess  of  the  lachrymal  sac.  The  B. 
coli  has  been  found  in  a  case  of  urethritis  (pseudo- 
gonorrhoea)  lying  inside  the  cells  like  gonococci,  and  it 
is  often  associated  with  the  pyogenic  cocci  in  cutaneous 
and  subcutaneous  purulent  inflammations. 

In  the  above-mentioned  diseases  the  colon  bacillus 
has  been  found  either  alone  or  associated  with  other 
pathogenic  bacteria  in  such  numbers  as  to  be  con- 
sidered a  factor  in  the  etiology  of  the  disease,  and  in 
some  cases  there  is  no  reason  to  doubt  that  it  is  the 
primary  cause  of  infection.  Though  further  study  and 
investigation  are  required  to  show  the  specific  patho- 
genic properties  of  this  micro-organism,  it  is  evident 


454  BACTERIOLOGY. 

that  under  certain  conditions  it  may  become  pathogenic 
to  man. 

According  to  many  authorities  there  are  a  great 
number  of  varieties  of  the  colon  bacillus,  some  main- 
taining even  that  it  may  become,  under  suitable  con- 
ditions, identical  with  the  typhoid  bacillus;  but  there 
has  been  no  proof  whatever  of  this. 

Differential  Diagnosis.  By  comparing  what  has  been 
said  of  the  bacillus  coli  and  the  bacillus  typhosus  it 
will  be  seen  that  while  certain  varieties  of  each  simu- 
late each  other  in  many  respects,  the  characteristic 
varieties  of  each  still  possess  individual  characteristics 
by  which  they  may  be  readily  differentiated: 

1.  The  motility  of  the  B.  coli  is,  as  a  rule,  much  less 
conspicuous  than  that  of  the  B.  typhosus.     It  is  also 
shorter,  thicker,  and  has  fewer  flagella. 

2.  In  gelatin  the  colonies  of  the  colon  bacillus  de- 
velop more  rapidly  and  luxuriantly  than  those  of  the 
typhoid  bacillus. 

3.  On  potato  the  growth  of   the  colon  bacillus  is 
usually  rapid,  luxuriant,  and  visible,  though  not  inva- 
riably so;  while  that  of  the  typhoid  bacillus  is  ordina- 
rily invisible. 

4.  The  colon  bacillus  coagulates  milk  in  from  thirty- 
six  to  forty-eight  hours  in   the  incubator,  with  acid 
reaction.    The  typhoid  bacillus  does  not  cause  coagula- 
tion. 

5.  The  colon  bacillus  is  conspicuous  for  its  power  of 
causing  fermentation,  with  the  production  of  gas  in 
media  containing  glucose.     The  typhoid  bacillus  never 
does  this. 

6.  In  nutrient  agar  or  gelatin  containing  lactose  and 
litmus  tincture,  and  of  a  slightly  alkaline  reaction,  the 


BACILLUS  CO  LI  COM  MUNIS.  455 

color  of  the  colonies  of  the  colon  bacillus  is  pink  and 
the  surrounding  medium  becomes  red;  while  the  colonies 
of  the  typhoid  bacillus  are  blue,  and  there  is  little  or  no 
reddening  of  the  surrounding  medium. 

7.  The  colon  bacillus  possesses  the  property  of  pro- 
ducing indol   in  cultures  of   bouillon  or  peptone;  the 
typhoid  bacillus  in  these   solutions  does  not  produce 
indol,  except  in  a  few  rare  exceptions. 

8.  The  colon    bacillus    rarely  produces  thread  out- 
growths in  the  Hiss  plate  medium.     The  typhoid  bacil- 
lus produces  thread  outgrowths  and  smaller  colonies  in 
this  medium.     In  the   Hiss   tube  medium  the   colon 
bacillus  produces  either  a  growth  limited  to  the  area 
inoculated  or  a  diffuse  growth  streaked  with  clear  lines 
and  spaces.     The  typhoid  bacillus  produces  a  diffuse 
growth  evenly  clouding  the  entire  medium. 

9.  On  the  Capaldi  medium  the  colon  colonies  are 
more  granular  and  darker  than  those  of  the  typhoid 
bacilli. 

10.  On  the  Eisner  medium  the  colon  colonies  appear 
earlier  and  become  larger  and  more  opaque  than  the 
average  typhoid  colonies. 

11.  Finally,  we  have  the  test  of  placing  the  bacilli 
in  animals  and  in  the  hanging  drop,  together  with  the 
serum  of  animals  immunized  to  either  the  colon  or  the 
typhoid  bacillus. 

None  of  these  tests  alone  can  be  depended  upon  for 
making  a  differential  diagnosis  of  the  colon  bacillus 
from  the  typhoid  bacillus  or  other  similar  bacilli. 

Unfortunately  in  most,  at  least,  of  these  characters 
certain  degrees  of  variation  may  often  be  observed  in 
different  cultures  of  the  typhoid  and  colon  bacillus 
which  may  lead  to  confusion.  For  instance,  the  mor- 


456  BACTERIOLOGY. 

phology  may  vary  considerably,  even  at  times  when 
grown  on  the  same  culture  media,  and  the  motility  is 
not  always  equally  active;  the  flagella  formation  may 
vary;  the  rapidity  of  growth  may  differ,  especially 
between  freshly  made  and  old  cultures;  the  grape-leaf 
appearance  of  the  surface  colonies  on  gelatin,  which  is 
usually  characteristic,  may  vary  with  the  composition 
of  the  gelatin,  at  times  no  typical  colonies  at  all  being 
presented;  the  threads  in  the  Hiss  media  may  be  lack- 
ing; the  indol  test  requires  great  care  in  its  perform- 
ance, and  in  rare  instances  the  typhoid  bacillus  produces 
it;  the  growth  on  potato  is  not  to  be  depended  on,  being 
often  visible  and  not  characteristic;  the  virulence  of 
both  the  bacilli  is  so  little  characteristic  that  it  can 
hardly  be  used  for  diagnostic  purposes;  and,  finally,  the 
serum  test  is  not  absolutely  infallible  in  all  cases,  for 
once  we  met  with  a  bacillus  in  feces  which  grew  in  a 
manner  utterly  at  variance  with  the  typhoid  bacillus, 
yet  still  gave  the  Widal  reaction  perfectly  with  the 
serum  of  an  immunized  horse.  It  is  also  stated  by 
Abbott  that  all  typhoid  bacilli  do  not  give  the  Widal 
reaction  with  the  serum  derived  from  a  typhoid  in- 
fection with  a  single  variety  of  a  typhoid  bacillus. 
This  is  an  experience  that  as  yet  we  have  not  met 
with.  The  Pfeiffer  reaction  in  guinea-pigs  is  a  matter 
of  extreme  delicacy,  aud  varying  results  are  sometimes 
obtained. 

In  spite,  however,  of  these  difficulties  it  is  very  easy 
to  sufficiently  identify  the  typhoid  bacillus  for  all  prac- 
tical purposes.  A  bacillus  which  grows  typically  in 
the  Hiss  tube  media  and  shows  the  Widal  reaction  with 
a  high  dilution  of  the  serum  of  an  animal  immunized 


BACILLUS  COLI  COMMUNIS.  457 

to  the  typhoid  bacillus,  is  in  all  probability  the  typhoid 
bacillus.  The  same  could  probably  be  stated  of  a 
bacillus  which  grew  characteristically  in  glucose  bouil- 
lon and  nutrient  gelatin,  and  also  showed  the  specific 
serum  reaction.  Probably  not  one  time  in  ten  thousand 
would  such  bacilli  prove  on  further  investigation  not 
to  be  typhoid  bacilli.  A  still  further  test  is  to  inocu- 
late animals  with  several  doses  of  the  dead  bacilli, 
whose  identification  is  sought,  and  note  whether  there 
is  produced  a  serum  which  agglutinates  typhoid  bacilli. 


CHAPTER   XXV. 

PNEUMOBACILLUS  ;     FRIEDLANDER' S    BACILLUS. 

DISCOVERED  by  Friedlander  (1883),  and  declared  by 
him  to  be  the  cause  of  fibrinous  pneumonia.  Subse- 
quent researches  have  shown  that  it  is  present  in  only 
a  small  proportion  of  the  cases  of  this  disease.  It  is 
found  also  not  infrequently  in  the  mucous  membranes 
of  the  mouth  and  air-passages  of  healthy  individuals, 
and  in  the  air. 

Morphology.  Short  bacilli  with  rounded  ends,  often 
resembling  micrococci,  especially  in  recent  cultures; 
commonly  united  in  pairs  or  in  chains  of  four,  and, 
under  certain  circumstances,  surrounded  by  a  trans- 
parent capsule.  This  capsule  is  not  seen  in  prepara- 
tions made  from  artificial  culture  media,  but  is  visible 
in  well-stained  preparations  from  the  blood  of  an  in- 
oculated animal. 

Friedlander' s  bacillus  stains  readily  with  the  aniline 
colors,  but  is  not  stained  by  Gram's  method. 

Biological  Characters.  An  aerobic,  non-motile,  non- 
liquefying  bacillus;  also  facultative  anaerobic;  does  not 
form  spores.  In  gelatin  stick  cultures  it  presents  the 
"  nail-shaped  "  growth  first  described  by  Friedlander, 
which  is  not,  however,  peculiar  to  this  bacillus.  Gas- 
bubbles  occasionally  develop  in  gelatin,  and  in  old 
cultures  the  gelatin  acquires  a  distinct  brownish  colora- 
tion. This  latter  characteristic  distinguishes  the  growth 


PNE  UMOBA  GILL  US.  459 

of  this  bacillus  from  that  of  the  bacillus  aerogenes, 
which  is  otherwise  very  similar  to  it  morphologically 
and  culturally.  On  gelatin  plates  colonies  appear  at  the 
end  of  twenty-four  hours  as  small  white  spheres,  which 
rapidly  increase  in  size.  These  colonies,  when  ex- 
amined by  a  low-power  lens,  present  a  somewhat 
irregular  outline  and  a  slightly  granular  appearance. 
The  growth  on  agar  is  in  quite  large  and  moist  grayish 
colonies.  On  blood-serum  abundant,  grayish-white, 
viscid  masses  are  developed.  The  growth  on  potato 
is  luxuriant — a  thick,  yellowish-white,  glistening  layer 
rapidly  covering  the  entire  surface.  Milk  is  not  coagu- 
lated. Indol  is  produced  in  bouillon  or  peptone  solu- 
tions. Fermentation  of  rnilk-sugar  and  glucose  is 
caused.  Growth  occurs  at  16°  to  20°  C.,  but  is  more 
rapid  at  37°  C. 

Pathogenesis.  Friedlander's  bacillus  is  pathogenic 
for  mice  and  guinea-pigs,  less  so  for  dogs,  and  rabbits 
are  apparently  immune.  In  Friedlander's  experiments 
mice  proved  to  be  particularly  susceptible.  These 
animals,  when  pure  cultures  of  the  bacillus  are  in- 
jected through  the  thoracic  wall  into  the  tissue  of  the 
lung,  invariably  succumb  to  the  disease.  On  autopsy 
the  pleural  cavities  are  found  to  contain  a  sero-puru- 
lent  fluid,  the  lungs  are  intensely  congested,  and  in 
places  show  limited  areas  of  red  hepatization;  the 
spleen  is  considerably  enlarged,  and  bacilli  are  present 
in  the  lungs,  the  pleuritic  fluid,  and  the  blood.  In 
guinea-pigs  which  are  killed  by  the  inoculation  similar 
appearances  are  observed. 

Friedlander's  bacillus  has  been  found  in  man,  not 
only  in  patients  suffering  from  croupous  pneumonia 
and  other  respiratory  diseases,  but  also  in  healthy  indi- 


460  BACTERIOLOGY. 

viduals,  and  in  the  outside  world.  Thus,  Pawlowsky 
found  it  in  the  atmosphere  and  Mori  in  canal  water; 
Netter  observed  it  in  4.5  per  cent,  of  the  cases  ex- 
amined by  him  in  the  saliva  of  healthy  individuals, 
and  Pansini  in  cases  of  pulmonary  tuberculosis  in 
the  sputum.  Friedlander  believed  that  the  bacillus 
described  by  him  was  the  specific  cause  of  croupous 
pneumonia;  but  in  129  cases  examined  by  Weichsel- 
baum  this  bacillus  was  found  in  only  9;  of  70  cases 
examined  by  Wolf  only  3  showed  the  presence  of  Fried- 
lander's  pneumobacillus.  It  is  evident,  therefore,  that 
though  this  micro-organism  may  be  concerned  in  the 
production  of  certain  forms  of  the  disease,  it  is  not  the 
specific  cause  of  croupous  pneumonia.  The  cases  which 
are  due  primarily  to  the  pneumobacillus  are  distin- 
guished, according  to  Weichselbaum  and  Netter,  by 
their  peculiarly  malignant  type  and  by  the  viscidity  of 
the  exudate  produced.  This  bacillus  is  also  probably 
concerned,  primarily  or  secondarily,  under  certain  cir- 
cumstances, in  the  production  of  pleurisy,  abscess  of 
the  lungs,  pericarditis,  endocarditis,  otitis  media,  and 
meningitis,  in  all  of  which  diseases  it  has  been  found 
at  times  to  be  present. 


CHAPTER   XXVI. 

THE    PRODUCERS   OF    ABSCESS,    CELLULITIS,    SEPTI- 
CAEMIA, ETC. 

THE  STAPHYLOCOCCI.     THE   MICROCOCCUS 
TETRAGENUS. 

The  Staphylococcus  Pyogenes  Aureus.     (The  Golden 
Staphylococcus.) 

THE  Staphylococcus  pyogenes  aureus  is  one  of  the 
commonest  pathogenic  bacteria,  being  almost  every- 
where present,  and  is  the  organism  most  frequently 
concerned  in  the  production  of  acute,  circumscribed, 
suppurative  inflammations.  It  was  first  observed  by 
Ogston  (1881)  in  the  pus  of  acute  abscesses,  but  was 
not  obtained  by  him  in  pure  culture.  It  was  isolated 
from  the  pus  of  acute  abscesses  and  accurately  described 
by  Rosenbach  (1884). 

Morphology.  Small,  spherical  cells,  having  a  diameter 
of  0.87/y.  (Passet),  occurring  solitary,  in  pairs  as  diplo- 
cocci,  in  short  chains  of  three  or  four  elements,  or  in 
groups  of  four,  but  most  commonly  in  irregular  masses, 
simulating  clusters  of  grapes ;  hence  the  name  staphylo- 
COOGUS.  (See  Fig.  59.) 

It  stains  quickly  in  aqueous  solutions  of  the  basic 
aniline  colors.  When  previously  stained  with  methyl- 
violet  it  is  not  decolorized  by  Gram's  method. 

Biology.  The  Staphylococcus  pyogenes  aureus  is  an 
aerobic,  facultative  anaerobic,  liquefying  micrococcus, 


462  BACTERIOLOGY. 

growing  readily  at  a  temperature  from  18°  to  20°  C., 
but  best  at  37°,  in  milk,  bouillon,  and  other  liquid 
media,  and  in  nutrient  gelatin  or  agar,  accompanied  by 
liquefaction  of  the  gelatin. 

Growth  on  Gelatin.  Grown  on  gelatin  plates  it 
develops,  at  room-temperature  within  forty-eight  hours, 
punctiform  colonies,  which,  when  examined  under  a 
low-power  lens,  appear  as  circular  disks  of  a  pale  brown 
color,  somewhat  darker  in  the  centre,  and  surrounded 
by  a  smooth  border.  The  colonies  grow  rapidly.  The 

FIG.  59. 


Staphylococcus.    X  1100  diameters. 

appearance  of  the  growth  is  most  characteristic.  Im- 
mediately surrounding  the  colonies,  which  are  of  a  pale 
yellow  color,  there  is  a  deepening  of  the  surface  of  the 
gelatin,  due  to  its  liquefaction.  By  suitable  light  a 
number  of  these  shallow  depressions  with  sharply  de- 
fined outlines  may  be  seen  on  the  gelatin  plate,  having 
a  diameter  of  from  5  to  10  mm.,  in  the  centres  of  which 
lie  the  yellow  colonies.  Later,  the  liquefaction  becomes 
general,  the  colonies  running  together.  In  stick  cul- 
tures in  gelatin  a  white  confluent  growth  at  first  appears 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.    463 

along  the  line  of  puncture,  followed  by  liquefaction  of 
the  medium,  which  rapidly  extends  to  the  sides  of  the 
test-tube.  At  the  end  of  two  days  the  yellow  pigmen- 
tation begins  to  form,  and  this  increases  in  intensity 
for  eight  days.  Finally,  the  gelatin  is  completely 
liquefied,  and  the  "  golden  staphylococci "  form  a 
golden-yellow  or  orange-colored  deposit  at  the  bottom 
of  the  tube.  Under  unfavorable  conditions  the  staphyl- 
ococcus  aureus  gradually  loses  its  ability  to  make  pig- 
ment and  to  liquefy  gelatin. 

Growth  on  Agar.  In  streak  and  stick  cultures  on 
agar  a  whitish  growth  is  at  first  produced,  and  this 
at  the  end  of  a  few  days  becomes  golden-yellow  on  the 
surface.  The  yellow  pigmentation  is  produced  only  in 
the  presence  of  oxygen;  colonies  found  at  the  bottom 
of  a  stab  culture  or  under  a  layer  of  oil  remain  white. 

Milk  inoculated  with  this  inicrococcus  at  the  end  of 
from  one  to  eight  days  is  coagulated;  bouillon  and 
peptone  solutions  are  densely  clouded  by  the  lux- 
uriant growth  produced. 

In  the  three  last-named  culture  media,  as  the  result 
of  the  growth  of  the  staphylococcus  aureus,  there  is  a 
production  of  acid  in  considerable  quantities,  these  con- 
sisting chiefly  of  lactic,  butyric,  a.nd  valerianic  acids. 
These  acids  have  been  supposed  to  play  a  part  in  the 
production  of  pus,  in  which,  according  to  some  ob- 
servers, they  are  often  present. 

The  staphylococcus  is  distinguished  from  most  other 
pathogenic  bacteria  by  its  comparatively  greater  power 
of  resistance  to  outside  influences,  desiccation,  etc.,  as 
well  as  to  chemical  disinfectants.  Cultures  of  the 
staphylococcus  pyogenes  in  gelatin  or  agar  retain  their 
vitality  for  a  year  or  more.  Its  thermal  death-point  is 


464  BACTERIOLOGY. 

between  56°  and  60°  0.,  the  time  of  exposure  being 
ten  minutes  (Sternberg).  Bolton  found  that  a  1  per 
cent,  solution  of  carbolic  acid  destroyed  the  vitality 
after  two  hours'  exposure.  Mercuric  chloride,  1  :  1000, 
destroys  it  in  from  five  to  ten  minutes,  according  to 
most  authorities,  though  Abbott  found  that  in  the 
same  culture  there  may  be  a  considerable  difference  in 
the  resisting  power  of  the  cocci,  all  being  frequently 
destroyed  in  five  minutes,  while,  again,  some  may  sur- 
vive after  an  exposure  to  a  solution  of  1  :  1000  for  ten, 
twenty,  and  even  thirty  minutes. 

Pathogenesis.  The  pathogenic  effect  of  the  staphylo- 
coccus  pyogenes  aureus  on  test  animals  varies  consider- 
ably according  to  the  mode  of  application  and  the 
virulence  of  the  special  culture  employed.  In  the  exper- 
iments so  far  made  this  micrococcus,  as  found  in  sup- 
purative  processes  in  the  human  subject,  has  not  proved 
to  be  as  infectious  for  animals  as  it  is  for  man.  In 
man  a  simple  rubbing  of  the  surface  of  the  unbroken  skin 
with  pus  from  an  acute  abscess  is,  as  a  rule,  sufficient 
to  produce  purulent  inflammation,  and  the  introduction 
of  a  few  germs  from  a  septic  case  into  a  wound  may  lead 
to  a  fatal  pysemia.  These  conditions  can  only  be  repro- 
duced in  lower  animajs  with  difficulty  and  by  the  inocu- 
lation of  large  quantities  of  the  culture.  Subcutaneous 
injections,  or  the  inoculation  of  open  wounds  in  mice, 
guinea-pigs,  and  rabbits,  are  commonly  without  result; 
occasionally  abscess  formation  may  follow  at  the  point 
of  inoculation,  which  usually  ends  in  recovery.  The 
pus-producing  property  of  the  organism  is  exhibited 
in  proportion  to  the  virulence  of  the  culture  employed. 
Slightly  virulent  cultures,  which  constitute  the  majority 
of  those  obtained  from  pus  taken  from  the  human  sub- 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.    465 

ject,  when  injected  subcutaneously  in  large  quantities 
(several  c.c.  of  a  fresh  bouillon  culture)  in  rabbits  or 
guinea-pigs,  give  rise  to  local  pathological  lesions — acute 
abscesses.  When  virulent  cultures  are  used — which  are 
rarely  obtainable — 0.5  c.c.  of  a  fresh  bouillon  culture 
is  sufficient  to  produce  similar  results.  The  abscesses 
heal  generally  without  treatment;  sometimes  the  ani- 
mals die  from  marasmus  in  consequence  of  the  sup- 
purative  process.  In  intraperitoneal  inoculations  the 
degree  of  virulence  of  the  culture  employed  is  still 
more  conspicuous  in  the  effects  produced.  The  ani- 
mals usually  die  in  from  two  to  nine  days.  The  most 
characteristic  pathological  lesions  are  found  in  the  kid- 
neys, which  contain  numerous  small  collections  of  pus, 
and  under  the  microscope  present  the  appearances 
resulting  from  embolic  nephritis.  Punctiform,  whitish- 
yellow  masses  of  the  size  of  a  pea  are  found  permeating 
the  pyramids.  Many  of  the  capillaries  and  some  of  the 
smaller  arteries  of  the  cortex  are  plugged  up  with 
thrombi  consisting  of  micrococci.  Metastatic  abscesses 
may  also  be  observed  in  the  joints  and  muscles.  The 
micrococci  may  be  recovered  in  pure  cultures  from  the 
blood  and  the  various  organs;  but  they  are  not  numer- 
ous in  the  blood  and  are  often  difficult  to  demonstrate 
microscopically.  Intravenous  inoculations  of  animals 
are  followed  by  similar  pathological  changes  Orth 
and  Wyssokowitsch  first  pointed  out  that  injection  of 
staphylococci  into  the  circulation,  after  injuring  the 
cardiac  valves  in  rabbits,  produced  ulcerative  endo- 
carditis. Subsequently,  Weichselbaum,  Prudden,  and 
Fraenkel  and  Sanger  obtained  confirmatory  results, 
thus  establishing  the  fact  that  when  the  valves  are  first 
injured,  mechanically  or  chemically,  the  injection  into  a 

30 


466  BACTERIOLOGY. 

vein  of  a  pure  culture  of  staphylococcus  aureus  gives 
rise  to  a  genuine  ulcerative  endocarditis.  It  has  been 
further  shown  by  Kibbert  that  the  same  result  may  be 
obtained  without  previous  injury  to  the  valves  by  in- 
jecting into  a  vein  the  staphylococcus  from  a  potato 
culture  suspended  in  water.  In  his  experiments  not 
only  the  micrococci  from  the  surface,  but  the  super- 
ficial layer  of  the  potato  was  scraped  off  with  a  steril- 
ized knife  and  mixed  with  distilled  water,  and  the 
successful  result  is  ascribed  to  the  fact  that  the  little 
agglomerations  of  micrococci  and  infected  fragments  of 
potato  attach  themselves  to  the  margins  of  the  valves 
more  readily  than  isolated  cocci  would  do.  Not  infre- 
quently, also,  in  intravenous  inoculations  of  young  ani- 
mals there  occurs  a  localization  of  the  injected  material 
in  the  marrow  of  the  small  bones.  This  may  take  place 
in  full-grown  animals  when  the  bones  have  been  injured 
or  fractured.  The  experimental  osteomyelitis  thus  pro- 
duced has  been  demonstrated  to  be  anatomically  analo- 
gous to  this  disease  in  man.  With  regard  to  the  lesions 
found  in  the  kidneys  after  intraperitoneal  or  intravenous 
inoculation  of  cultures  of  the  staphylococcus,  it  has  been 
found  that  when  injected  in  considerable  quantities  the 
organism  may  be  obtained  in  cultures  from  the  urine, 
but  not  sooner  than  six  or  eight  hours  after  the  injec- 
tion, and  not  until  the  formation  of  purulent  foci  in 
the  kidneys  has  already  occurred. 

The  Production  of  Toxic  Substances.  The  peculiar 
energetic  action  of  the  staphylococcus  pyogenes  aureus 
on  the  tissues  of  warm-blooded  animals  would  seem 
to  indicate  that  toxic  substances  are  produced  by 
this  organism,  which  play  an  important  part  in  its 
infective  properties.  Grawitz  and  De  Bary  have 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.     467 

shown  by  experiments  that  cultures  of  the  staphylo- 
coccus, when  sterilized  by  boiling  and  injected  subcuta- 
neously  into  dogs,  will  produce  local  abscesses.  Leber 
found  also  that  sterilized  cultures  introduced  into  the 
anterior  chamber  of  the  rabbit's  eye  would  bring  about 
a  fibro-purulent  inflammation,  the  cornea  becoming  in- 
sensible, and  perforation  alongside  of  the  sclerotic  ring 
finally  taking  place,  followed  by  the  formation  of  pus 
in  the  anterior  chamber  and  recovery.  These  local 
changes  are  the  results  of  the  inoculation  of  small  quan- 
tities only  of  the  dead  cultures;  but  when  large  amounts 
are  injected  into  a  vein  or  into  the  abdominal  cavity, 
toxic  effects  are  produced.  Dogs  and  guinea-pigs  thus 
treated  usually  die,  showing  symptoms  of  poisoning. 
From  the  bodies  of  the  bacteria  Leber  obtained,  by 
treating  them  with  alcohol  and  ether,  a  crystalline, 
chemical  substance,  which  he  called  phlogosin.  This 
substance,  which  is  an  energetic  pus-producer,  is  sup- 
posed to  be  the  active  principle  of  the  staphylococcus 
aureus. 

Immunization.  Immunity  against  staphylococcus  in- 
fection may  be  produced  in  different  animal  species 
by  the  injection  of  increasing  doses  of  the  pure  culture, 
either  living  or  previously  sterilized  by  boiling.  Reichel 
thus  succeeded  in  immunizing  dogs  against  a  surely 
fatal  dose  of  living  as  well  as  dead  staphylococci. 
Viquerat  claims  to  have  immunized  horses  in  the  same 
way. 

The  blood-serum  of  animals  which  have  been  im- 
munized by  means  of  living  or  dead  cultures  possesses 
slight  immunizing  and  curative  effects  in  other  animals, 
but  no  practical  use  of  the  serum  has  been  attempted 
in  man. 


468  BACTERIOLOGY. 

Occurrence  in  Man.  The  staphylococcus  pyogenes 
aureus  is  the  commonest  pyogenic  micro-organism  found 
in  man.  From  the  fact  that  these  micrococci  are  so 
constantly  present  in  the  pus  of  acute  abscesses,  as  de- 
monstrated by  Ogston,  Rosenbach,  Passet  and  others, 
it  was  formerly  assumed  that  there  could  be  no  pus- 
formation  in  the  absence  of  micro-organisms  of  this 
class;  but  it  is  now  well  known,  from  the  experiments, 
that  certain  chemical  substances,  such  as  nitrate  of 
silver,  oil  of  turpentine,  strong  liquor  ammonise,  etc., 
introduced  beneath  the  skin,  give  rise  to  pus-formation 
quite  independently  of  bacteria.  Practically  all  micro- 
organisms, moreover,  have  been  shown  by  experiment  to 
produce  under  certain  conditions  the  formation  of  pus 
by  their  products  when  inoculated  into  the  animal  body ; 
but,  while  this  has  been  demonstrated,  the  extended 
researches  of  bacteriologists  show  that  few  species  are 
usually  concerned  in  the  production  of  acute  abscesses, 
furuncles,  etc.,  in  man.  Of  these  the  two  most  im- 
portant, by  reason  of  their  frequent  occurrence  and 
pathogenic  power,  are  staphylococcus  pyogenes  aureus 
and  streptococcus  pyogenes;  next  to  these  comes  the 
staphylococcus  pyogenes  albus.  Two  or  more  species 
are  often  found  in  the  same  abscess;  thus,  Passet, 
in  33  cases  of  acute  abscess,  found  staphylococcus 
aureus  and  albus  associated  in  11,  albus  alone  in  4, 
albus  and  citreus  in  2,  streptococcus  pyogenes  alone  in 
8,  albus  and  streptococcus  in  1,  and  albus,  citreus,  and 
streptococcus  in  1. 

As  the  result  of  extended  researches,  however,  made 
by  bacteriologists  within  recent  years  the  golden  staphy- 
lococcus has  been  demonstrated  not  only  in  furuncles 
and  carbuncles,  but  also  in  various  pustular  affections 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.    469 

of  the  skin  and  mucous  membranes — impetigo,  sycosis, 
phlyctenular  conjunctivitis;  in  purlent  conjunctivitis 
and  inflammation  of  the  lachrymal  sac;  in  acute  ab- 
scesses formed  in  the  lymphatic  glands,  the  parotid 
gland,  the  tonsils,  the  mammae,  etc.;  in  metastatic 
abscesses  and  purulent  collections  in  the  joints;  in  em- 
pyema;  in  infectious  osteomyelitis,  in  ulcerative  endo- 
carditis, pyelonephritis,  etc.  It  is  one  of  the  chief 
etiological  factors  in  the  production  of  pyaemia  in  the 
various  pathological  forms  of  that  condition  of  disease. 

Not  all  persons  are  equally  susceptible  to  infection 
by  the  staphylococcus ;  those  who  are  in  a  cachectic 
condition  or  suffering  from  constitutional  diseases,  like 
diabetes,  are  especially  predisposed  to  infection.  In 
healthy  individuals  certain  parts  of  the  body,  as  the 
back  of  the  neck  and  the  seat,  are  more  liable  to  be 
attacked  than  others,  with  the  production  of  furuncles, 
carbuncles,  etc.  In  persons  in  whom  sores  are  readily 
caused,  in  consequence  of  disturbances  of  nutrition,  as 
in  exhausting  diseases,  the  micrococci  settle  at  the  points 
of  least  resistance.  Such  conditions  are  present  in  the 
bones  of  debilitated  young  children,  in  fractures,  and 
injuries  in  general. 

The  pyogenic  properties  of  the  staphylococcus  have 
been  demonstrated  upon  man  by  numerous  experiments. 
Garre  inoculated  a  small  wound  at  the  edge  of  one  of 
his  finger-nails  with  a  minute  quantity  of  a  pure  cul- 
ture, and  purulent  inflammation  extending  around  the 
margin  of  the  nail  resulted  from  the  inoculation. 
Staphylococcus  aureus  was  recovered  in  cultures  from 
the  pus  thus  formed.  The  same  observer  applied  a 
considerable  quantity  of  a  pure  culture  obtained  from 
this  pus — third  generation — to  the  unbroken  skin  of 


470  BACTERIOLOGY. 

his  forearm,  rubbing  it  well  into  the  skin.  At  the  end 
of  four  days  a  large  carbuncle,  surrounded  by  isolated 
furuncles,  developed  at  the  point  where  the  culture  had 
been  applied.  This  ran  the  usual  course,  and  it  was 
several  weeks  in  healing.  No  less  than  seventeen  scars 
remained  to  testify  to  the  success  of  the  experiment. 
Bockhart  rubbed  upon  the  uninjured  skin  of  the  fore- 
arm a  small  quantity  of  an  agar  culture  suspended  in 
salt  solution.  By  gently  scratching  with  a  disinfected 
finger-nail  the  epithelium  was  removed  in  places  over 
the  area  to  which  the  micrococcus  had  been  applied. 
Numerous  impetigo  pustules,  and  occasionally  a  gen- 
uine furuncle,  developed  as  the  result  of  the  procedure. 
Bockhart  examined  portions  of  the  skin,  which  he  ex- 
cised for  the  purpose,  under  the  microscope,  and  came 
to  the  conclusion  that  the  cocci  penetrate  by  way  of  the 
hair-follicles,  the  sebaceous  and  sudoriparous  glands,  or, 
where  the  epidermis  had  been  removed  by  scratching, 
directly  to  the  deeper  Jayers  of  the  skin. 

Staphylococcus  Pyogenes  Albus. 

Isolated  by  Rosenbach  (1884)  from  the  pus  of  acute 
abscesses,  in  which  it  is  sometimes  the  only  micro- 
organism present,  and  sometimes  associated  with  the 
staphylococcus  aureus  and  other  pyogenic  cocci. 

It  is  morphologically  identical  with  the  staphylococcus 
pyogenes  aureus,  and  is  probably  the  same  organism, 
which  has  lost  the  property  of  producing  pigment. 
On  the  average  it  is  somewhat  less  pathogenic.  The 
surface  cultures  upon  nutrient  agar  and  potato  have  a 
milk-white  color.  Its  biological  characters  are  not  to 
be  distinguished  from  the  staphylococcus  aureus. 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.    471 

According  to  Passet,  it  is  more  common  than  the 
aureus  in  man;  but  the  majority  of  bacteriologists 
agree  with  Rosenbach,  that  the  aureus  is  found  at 
least  twice  as  frequently  in  human  pathological  pro- 
cesses as  the  albus. 

Staphylococcus  Epidermis  Albus  (Welch). 

Probably  identical  with  the  staphylococcus  pyogenes 
albus,  but  found  by  Welch  on  the  surface  of  the  body, 
though  often  present  in  parts  of  the  epidermis  deeper 
than  can  be  reached  by  any  known  means  of  cutaneous 
disinfection  save  the  application  of  heat. 

With  reference  to  this  micrococcus,  Welch  says : 
"  So  far  as  our  observations  extend — and  already  they 
amount  to  a  large  number — this  coccus  may  be  regarded 
as  a  nearly,  if  not  quite,  constant  inhabitant  of  the 
epidermis.  It  is  now  clear  why  I  have  proposed  to 
call  it  the  staphylococcus  epidermis  albus.  It  possesses 
such  feeble  pyogenic  capacity,  as  is  shown  by  its  be- 
havior in  wounds,  as  well  as  by  experiments  on  rab- 
bits, that  the  designation  staphylococcus  pyogenes  albus 
does  not  seem  appropriate.  Still,  I  am  not  inclined  to 
insist  too  much  upon  this  point,  as  very  probably  this 
coccus,  which  has  hitherto  been  unquestionably  identi- 
fied by  Bossowski  and  others  with  the  ordinary  staphylo- 
coccus pyogenes  albus  of  Rosenbach,  is  an  attenuated 
or  modified  form  of  the  latter  organism,  although,  as 
already  mentioned,  it  presents  some  points  of  difference 
from  the  classical  description  of  the  white  pyogenic 
coccus  ." 

• 

According  to  Welch,  this  coccus  differs  from  the 
staphylococcus  aureus  not  only  in  color,  but  also  in 
the  fact  that  it  liquefies  gelatin  more  slowly,  does  not  so 


472  BACTERIOLOGY. 

quickly  cause  coagulation  in  milk,  and  is  far  less  viru- 
lent when  injected  into  the  circulation  of  rabbits.  It 
has  been  shown  by  the  experiments  of  Bossowski  and 
of  Welch  that  this  micro-organism  is  very  frequently 
present  in  aseptic  wounds,  and  that  usually  it  does 
not  materially  interfere  with  the  healing  of  wounds, 
although  sometimes  it  appears  to  cause  suppuration 
along  the  drainage-tube,  and  it  is  the  common  cause 
of  "  stitch  abscess." 

Staphylococcus  Pyogenes  Citreus. 

Isolated  by  Passet  (1885)  from  the  pus  of  acute 
abscesses,  in  which  it  is  occasionally  found  (about  10 
per  cent,  of  the  cases  examined)  in  association  with 
other  pyogenic  cocci.  It  is  morphologically  identical 
with  the  Staphylococcus  aureus  and  albus,  being  distin- 
guished from  the  other  species  only  by  the  formation  of 
a  lemon-yellow  pigment  instead  of  a  golden-yellow,  as 
in  the  aureus,  and  a  white  or  colorless  deposit,  as  in  the 
albus. 

THE  MICROCOCCUS  TETRAGENUS. 

This  organism  was  discovered  by  Gaffky  (1881).  It 
is  not  infrequently  present  in  the  saliva  of  healthy  in- 
dividuals and  in  the  sputum  of  consumptive  patients. 
In  sputum  it  is  sometimes  an  evidence  of  mouth  con- 
tamination rather  than  lung  infection.  It  has  repeatedly 
been  observed  in  the  walls  of  cavities  in  pulmonary 
tuberculosis  associated  with  other  pathogenic  bacteria, 
which,  though  playing  no  part  in  the  etiology  of  the 
original  disease,  contribute,  doubtless,  to  the  progressive 
destruction  of  the  lung.  Its  pyogenic  character  is  shown 
by  its  occasional  occurrence  in  the  pus  of  acute  ab- 


PRODUCERS  OF  ABSCESS,   CELLULITIS,  ETC.     473 

scesses.     Its  presence  has  also  been  noted  in  the  pus  of 
empyema  following  pneumonia. 

Morphology.  Micrococci  having  a  diameter  of  about 
Ijut,  which  divide  in  two  directions,  forming  tetrads, 
and  bound  together  by  a  transparent,  gelatinous  sub- 
stance, enclosing  the  cell  like  a  capsule.  In  cultures 
the  cocci  are  seen  in  various  stages  of  division  as  large, 
round,  undivided  cells,  in  pairs  of  oval  elements,  and 
in  groups  of  three  and  four  (Fig.  60).  When  the  divis- 

PlG.  60. 


Micrococcus  tetragenus.    X  1000  diameters. 

ion  is  complete  they  remind  one  of  sarcinae  in  appear- 
ance, except  that  they  do  not  divide  in  three  directions 
and  are  not  built  up  like  diminutive  cotton  bales. 

This  micrococcus  stains  readily  with  the  ordinary  ani- 
line dyes;  the  transparent  gelatinous  envelope  is  only 
feebly  stained.  It  is  not  decolorized  by  Gram's  method. 

Biological  Characters.  The  growth  of  this  micro- 
coccus  is  slow  under  all  conditions.  It  grows  both  in 
the  presence  and  absence  of  oxygen;  it  grows  best  from 
35°  to  38°  C.,  but  may  be  cultivated  also  at  the  ordi- 
nary room-temperature — about  20°  C. 


474  BA  GTERIOL  OGY. 

Growth  on  Gelatin.  On  gelatin  plates  small,  white 
colonies  are  developed  in  from  twenty-four  to  forty- 
eight  hours,  which,  when  examined  under  a  low- 
power  lens,  are  seen  to  be  spherical  or  lemon-shaped, 
grayish-yellow  disks,  with  a  finely  granular  or  mul- 
berry-like surface,  and  a  uniform,  but  somewhat  roughly 
dentated  border.  When  the  colonies  push  forward  to 
the  surface  of  the  gelatin  they  form  white,  elevated, 
drop-like  masses,  having  a  diameter  of  1  to  2  mm.  In 
gelatin  stick  cultures  the  colonies  may  be  either  isolated 
or  confluent,  in  the  case  forming  a  thick,  white,  slimy 
mass,  filling  out  the  fissures  and  hollow  spaces  all 
along  the  line  of  puncture;  on  the  surface  a  broad, 
thick  layer  of  4  to  5  mm.  in  extent  is  apparent.  The 
gelatin  is  not  liquefied. 

Growth  on  Agar  and  Blood-serum.  On  plate  and 
slant  cultures  of  agar  and  blood-serum  the  surface 
of  the  growth  is  moist  and  glistening.  The  colonies 
appear  as  small,  transparent,  round  points,  which  have 
a  grayish-yellow  color  and  are  slightly  elevated  above 
the  surface  of  the  medium. 

Pathogenesis.  Subcutaneous  injections  of  a  culture 
of  this  micrococcus  in  minute  quantity  is  usually  fatal 
to  white  mice.  The  animals  remain  apparently  well 
for  a  day  or  two,  then  become  quiet,  until  death  takes 
place  on  the  third  or  sixth  day.  The  micrococci  are 
found  in  comparatively  small  numbers  in  the  blood  of 
the  vessels  and  heart,  but  are  more  numerous  in  the 
spleen,  lungs,  liver,  and  kidneys.  Gray  mice  are,  for 
the  most  part,  immune  to  infection  by  the  micrococcus 
tetragenus.  Guinea-pigs  at  times  show  only  a  local 
reaction  after  inoculation,  and  again  die  from  septi- 
csemic  infection.  When  intraperitoneal  injections  are 


PRODUCERS  OF  ABSCESS,  CELLULITIS,  ETC.    475 

given  they  are  followed  by  purulent  peritonitis,  beau- 
tifully formed  cocci  in  groups  of  four  being  obtained 
in  immense  numbers  in  the  exudate.  Rabbits  and  dogs 
are  not  affected  by  large  doses  of  a  culture  subcuta- 
neously  or  intravenously  administered. 

The  serum  from  immunized  cases  has  not  been  used 
therapeutically  in  human  infection. 


CHAPTER  XXVII. 

STREPTOCOCCUS  PYOGENES  (STREPTOCOCCUS  ERYSIPE- 
LATUS  ;  STREPTOCOCCUS  OF  PUS  ;  STREPTOCOCCUS 
PATHOGENES  LONGUS). 

THIS  micrococcus  was  first  observed  by  Koch  in 
stained  sections  of  tissues  attacked  by  septic  processes, 
and  by  Ogston  in  the  pus  of  acute  abscesses  (1882). 
It  was  obtained  by  Fehleisen  (1883)  in  pure  cultures 
from  a  case  of  erysipelas,  its  cultural  and  pathological 
characters  studied  and  demonstrated  by  him  to  be 
capable  of  producing  erysipelas  in  man.  Rosenbach 
(1884)  and  Krause  and  Passet  (1885)  isolated  the 
streptococcus  from  the  pus  of  acute  abscesses  and  gave 
it  the  name  of  streptococcus  pyogenes.  It  has  since 
been  proved  to  be  one  of  the  chief  etiological  factors 
in  the  production  of  many  suppurative  inflammations. 
Formerly  the  streptococci  of  erysipelas,  acute  abscesses, 
septicaemia,  puerperal  fever,  etc.,  were  thought  to  be- 
long to  different  species,  because  they  were  observed 
to  possess  apparent  differences  in  their  biological  and 
pathological  characteristics,  according  to  the  source 
from  which  they  were  obtained.  Thus  one  species  of 
streptococcus  was  believed  to  be  capable  of  causing 
erysipelas  only,  another  only  acute  abscesses,  another 
sepsis,  etc.;  but  it  is  now  known  that  the  slight  differ- 
ences between  the  majority  of  the  streptococci  growing 
in  long  chains  are  but  variations  of  one  and  the  same 


STREPTOCOCCUS  PTOGENES. 


477 


species  which  has  been  appropriately  termed  the  "strep- 
tococcus pathogenes  longus."  Some  of  the  streptococci, 
at  least  in  so  far  as  their  specific  products  and  their 
reaction  in  the  presence  of  a  curative  serum  is  con- 
cerned, belong  to  a  species  as  distinct  from  the  strepto- 
coccus pyogenes  as  the  pneumococcus.  This  question 
has  a  very  practical  side,  for  upon  its  decision  rests  our 
ability  to  choose  a  suitable  protective  serum  in  cases  of 
streptococcus  infection. 


FIG.  61. 


FIG.  62. 


Streptococci   in   peritoneal   fluid, 
partly  enclosed  in  leucocytes.     X 
1000  diameters. 


Streptococci  in  throat  exudate  smeared 
on  cover-glass.    X  1000  diameters. 


Morphology.  Spherical  cocci,  when  fully  developed, 
having  no  independent  movements,  from  0.4//  to  I/*  in 
diameter,  usually  larger  than  the  staphylococci,  but 
varying  in  dimensions  in  different  cultures  and  even  in 
different  parts  of  a  single  colony.  They  multiply  by 
binary  division  in  one  direction  only,  forming  chains  of 
eight,  ten,  twenty,  and  more  elements,  being,  however, 
often  associated  distinctly  in  pairs.  On  certain  media 
the  cocci  occur  mostly  in  diplococci,  but  usually  they 
grow  in  longer  or  shorter  chains.  Certain  cocci  fre- 


478  BACTERIOLOGY. 

quently  exceed  their  fellows  greatly  in  size,  especially 
in  old  cultures,  when  this  may  be  considered  to  be  the 
result  of  involution  forms.  (See  Figs.  61,  62,  63,  and 
64.) 

They  stain  readily  by  aniline  colors  and  by  Gram's 
method. 

FIG.  63.  FIG.  64. 


?TT> 

T«#\v?<& 

..'jj^fv^,* 

v^^i^'l 


Streptococci  from  solidified  serum  cul-  Streptococcus  growing  in  long 
ture  appearing  mostly  as  diplococci.  chains  in  bouillon  culture.  X  1000 
X  1000  diameters.  diameters. 

Biological  Characters.  Streptococci  grow  readily  in 
various  liquid  and  solid  culture  media.  The  most 
favorable  temperature  for  their  development  is  from 
30°  to  37°  C.,  but  they  multiply  freely  at  ordinary 
room- temperature — 18°  to  20°  C.  They  are  faculta- 
tive anaerobes,  growing  both  in  the  presence  and  ab- 
sence of  oxygen. 

Growth  on  Gelatin.  Tubes  of  gelatin  which  have 
been  inoculated  with  streptococci  by  puncture  with  the 
platinum  needle  show  on  the  surface  no  growth  beyond 
the  point  of  entrance.  In  the  depth  of  the  gelatin  on 
the  second  or  third  day  a  distinct,  tiny  band  appears, 


STREPTOCOCCUS  PYOGENES.  479 

with  granular  edges  or  made  up  of  granules.  These 
granules  may  be  very  fine  or  fairly  coarse.  They  are 
nearly  translucent,  with  a  whitish,  yellowish,  or  brown- 
ish tinge.  With  characteristic  cultures  the  gelatin  is 
not  liquefied,  though  occasionally,  with  unusual  varie- 
ties, a  certain  amount  of  liquefaction  has  been  observed 
to  take  place. 

Growth  on  Agar.  On  agar  plates  the  colonies  are 
visible  after  twelve  to  thirty  hours'  growth,  and  present 
a  beautiful  appearance  when  magnified  sufficiently  to 
see  the  individual  cocci  in  the  chain.  The  colonies 
from  different  sources  vary  in  size,  thickness,  mottling, 
color,  and  in  the  appearance  of  their  borders.  The 
streptococcus  growing  in  short  chains  in  bouillon  shows 
but  little  tendency  to  form  true  loops,  but  rather  pro- 
jecting rows  at  the  edges  of  the  colonies,  while  those 
growing  in  long  chains  show  beautiful  loops,  which  are 
characteristic  of  this  organism.  The  colonies  are  nearly 
circular  in  shape  when  thinly  scattered  over  the  plates, 
but  irregular  in  form  when  crowded  together. 

Growth  in  Bouillon.  Streptococci  grow  readily  in 
slightly  alkaline  bouillon  at  37°  G.,  reaching  their 
full  development  within  thirty-six  to  forty-eight  hours. 
Those  which  grow  in  long  chains  usually  give  an  abun- 
dant flocculent  deposit  and  leave  the  liquid  clear.  The 
deposit  may  be  in  grains,  in  tiny  flocculi,  in  larger 
flakes,  or  in  tough,  almost  membranous  masses,  the 
differences  depending  on  the  strength  of  union  between 
the  pairs  of  cocci  in  the  chains.  Some  of  the  strepto- 
cocci growing  in  long  chains,  however,  cause  the  broth 
to  become  cloudy.  This  cloudiness  may  be  only  tem- 
porary or  it  may  be  lasting.  Those  growing  in  short 
chains,  as  a  rule,  cloud  the  broth;  this  cloudiness 


480  BACTERIOLOGY. 

remaining  for  days  or  weeks.  A  granular  deposit 
appears  at  the  bottom  of  the  tube. 

The  development  in  a  mixture  of  ascitic  fluid  and 
bouillon,  which  is  the  best  medium  for  the  growth  of 
the  streptococcus,  is  more  abundant  than  in  bouillon. 
The  liquid  is  clouded,  and  a  precipitate  only  occurs 
after  some  days,  the  fluid  gradually  clearing. 

Growth  on  Solidified  Blood-serum.  This  is  also  an 
excellent  medium  for  the  streptococcus.  Tiny,  grayish 
colonies  appear  twelve  to  eighteen  hours  after  inocu- 
lation. 

Growth  in  Milk.  All  streptococci  grow  well  in  milk. 
As  a  rule,  coagulation  of  the  casein  occurs  with  the 
production  of  acid,  but  this  is  not  always  the  case. 

The  Duration  of  the  Life  of  Streptococci  Outside  of 
the  Body.  This  is  not,  as  a  rule,  very  great.  When 
dried  in  blood  or  pus,  however,  they  may  live  for 
several  months  at  room-temperature,  and  longer  in  an 
ice-chest;  and  in  gelatin  and  agar  cultures  they  live 
for  from  one  week  to  three  months;  in  bouillon  cult- 
ures they  are  usually  short-lived,  the  majority  dying 
within  two  or  three  days,  and  very  few  living  over  a 
month;  but  in  serum  bouillon  they  live  much  longer. 
In  order  to  keep  streptococci  alive  and  virulent,  it  is 
best  to  keep  them  in  serum  or  ascitic  fluid  bouillon  in 
small,  sealed  glass  tubes  in  the  ice-chest.  The  thermal 
death-point  of  the  streptococcus,  according  to  Stern- 
berg,  is  between  52°  and  54°  C.,  the  time  of  exposure 
being  ten  minutes. 

Von  Lingelsheim  has  reported  the  following  results 
obtained  in  an  extended  series  of  experiments  made  to 
determine  the  germicidal  power  of  various  chemical 
agents  as  tested  upon  this  micro-organism  (time  of  ex- 


STREPTOCOCCUS  PYOGENES.  481 

posure,  two  hours):  Mercuric  chloride,  1  :  2500;  sul- 
phate of  copper,  1  :  200;  trichloride  of  iodine,  1  :  750; 
peroxide  of  hydrogen,  i  :  50;  carbolic  acid,  1  :  300; 
cresol,  1  :  250;  lysol,  1  :  300;  creolin,  1  :  130. 

Pathogenesis.  The  majority  of  test  animals  are  not 
very  susceptible  to  infection  by  the  streptococcus,  and, 
hence,  it  is  difficult  to  obtain  any  definite  pathological 
alterations  in  their  tissues  through  the  inoculation  into 
them  of  cultures  of  this  organism  by  any  of  the  methods 
ordinarily  practised.  White  mice  and  rabbits,  under 
similar  conditions,  are  the  most  susceptible,  and  these 
animals  are,  therefore,  usually  employed  for  experimen- 
tation. Streptococci,  however,  differ  greatly  in  the 
effects  which  they  produce  in  inoculated  animals, 
according  to  their  animal  virulence,  which  is  very 
different  from  human  virulence.  The  most  virulent, 
when  injected  in  the  minutest  quantity  into  the  circu- 
lation or  into  the  subcutaneous  tissues  of  a  mouse  or 
rabbit,  produce  death  by  septicaemia.  Those  of  some- 
what less  virulence  produce  the  same  result  when  in- 
jected in  considerable  quantities.  Those  still  less  patho- 
genic produce  septicaemia,  which  may  be  mild  or  severe, 
when  injected  into  the  circulation;  but  when  injected 
subcutaneously,  they  produce  abscess  or  erysipelas. 
The  remaining  streptococci,  unless  introduced  in  quan- 
tities of  20  c.c.  or  over,  produce  only  a  slight  redness, 
or  no  reaction  at  all,  when  injected  subcutaneously, 
and  little  or  no  effect  when  injected  directly  into  the 
circulation.  Many  of  the  streptococci  obtained  from 
cases  of  cellulitis,  abscess,  empyema,  and  even  septi- 
caemia belong  to  this  group. 

A  number  of  varieties  of  streptococci  have  thus  been 
discovered,  differing  in  virulence  and  in  their  growth 

31 


482  BACTERIOLOGY. 

on  artificial  media;  but  all  attempts  to  separate  them 
into  various  classes,  until  recently  through  the  use  of 
specific  serum,  have  failed,  because  the  differences  ob- 
served, though  often  marked,  are  not  constant,  many 
varieties  having  been  found  to  lose  their  distinctive 
characteristics,  and  even  to  apparently  change  from  one 
class  to  another.  A  further  objection  to  any  previous 
classification  of  streptococci,  based  on  the  manner  of 
growth  on  artificial  culture  media,  is  that  it  has  been 
impossible  to  make  any  which  would  at  the  same  time 
give  even  an  approximate  idea  of  their  virulence.  Ex- 
periments have  proved  that  the  streptococci  originally 
virulent  may  become  non-virulent  after  long  cultivation 
on  artificial  media,  and,  again,  that  they  may  return  to 
their  original  properties  after  being  passed  through  the 
bodies  of  susceptible  animals.  The  peculiar  type  of 
virulence  which  they  may  acquire  tends  to  perpetuate 
itself,  at  least  for  a  considerable  time. 

One  important  fact  that  experience  teaches  us  is, 
that  those  streptococci  are  the  most  dangerous  to  any 
animal  which  have  come  immediately  from  septic  con- 
ditions in  the  same  species  of  animal,  and  the  more 
virulent  the  case  the  more  virulent  the  streptococci  are 
apt  to  be  in  other  animals  of  the  same  species.  There 
seems  also  to  be  a  strong  tendency  for  a  streptococcus 
to  produce  the  same  inflammation,  when  inoculated,  as 
the  one  from  which  it  was  obtained;  for  example,  strep- 
tococci from  erysipelas  tend  to  produce  erysipelas,  from 
septicaemia  to  produce  septicaemia,  etc.  Streptococci, 
however,  obtained  from  different  sources  (abscesses, 
puerperal  fever,  sepsis,  erysipelas,  etc.)  are  in  many 
instances  capable,  under  favorable  conditions,  of  pro- 
ducing erysipelas  when  inoculated  into  the  ear  of  a 


STREPTOCOCCUS  PYOGENES.  483 

rabbit,  as  has  been  proved  by  experiment,  provided 
they  possess  sufficient  virulence  (Knorr,  Petruschky). 
If  the  culture  does  not  have  the  required  virulence  to 
produce  this  effect  the  virulence  can  usually  be  acquired 
by  passage  through  animals.  Streptococci  obtained  from 
the  same  disease  and  from  the  same  individual  usually 
show  very  much  the  same  degree  of  virulence. 

Occurrence  in  Man.  Streptococci  have  been  found 
in  man  as  the  primary  cause  of  infection  in  the  follow- 
ing diseases :  Erysipelas,  acute  abscesses,  small  and 
large,  cellulitis,  circumscribed  as  well  as  diffused,  sep- 
sis, puerperal  infection,  lymphatic  abscesses,  angina, 
pneumonia,  periostitis,  otitis  media,  mastoiditis,  men- 
ingitis, empyema,  and  endocarditis.  Associated  with 
other  bacteria  in  diseases  of  which  they  were  the  specific 
cause,  they  have  also  been  found  as  the  secondary  or 
mixed  infection  in  many  diseases,  such  as  in  pulmonary 
tuberculosis,  bronchopneumonia,  septic  diphtheria,  and 
diphtheritic  scarlatina.  In  diphtheritic  false  membranes 
this  micrococcus  is  very  commonly  present,  and  is  fre- 
quently the  source  of  deeper  infection,  such  as  abscesses 
and  septicaemia;  and  in  certain  cases  attended  with  a 
diphtheritic  exudation,  in  which  the  Loffler  bacillus 
has  not  been  found  by  competent  bacteriologists,  it 
seems  probable  that  the  streptococcus  pyogenes,  alone 
or  with  other  pyogenic  cocci,  is  responsible  for  the  local 
inflammation  and  its  results.  These  forms  of  so-called 
diphtheria,  as  first  pointed  out  by  Prudden,  are  most 
commonly  associated  with  scarlatina  and  measles,  ery- 
sipelas, and  phlegrnonous  inflammation,  or  occur  in  in- 
dividuals exposed  to  these  or  other  infectious  diseases. 
So  uniformly  are  streptococci  present  in  the  pseudo- 
membranous  inflammations  of  patients  sick  with  scarlet 


484  BACTERIOLOGY. 

fever  that  many  investigators  have  suspected  them  to 
be  the  cause  of  this  disease  (Kurth,  Baginsky,  E-oskin). 
They  are  found,  however,  regularly  in  the  secretion  of 
healthy  individuals  (in  100  examinations  by  us  we  found 
them  in  83,  and  probably  could  have  found  them  in 
the  others  by  longer  search).  Their  presence  in  scarlet 
fever  is  most  probably  due  to  their  increase  in  the  dis- 
ordered mucous  membrane. 

The  causal  relation  of  the  streptococcus  to  the  above- 
mentioned  diseases  has  been  amply  proved  by  inocula- 
tion experiments  both  in  man  and  animals.  Fehleisen 
has  inoculated  cultures,  obtained  in  the  first  instance 
from  the  skin  of  patients  with  erysipelas,  into  patients 
in  the  hospital  suffering  from  inoperable  malignant 
growths — lupus,  carcinoma,  and  sarcoma — and  has 
obtained  positive  results,  a  typical  erysipelatous  in- 
flammation having  developed  around  the  point  of 
inoculation  after  a  period  of  incubation  of  from  fifteen 
to  sixty  hours.  This  was  attended  with  chilly  sensa- 
tions and  an  elevation  of  temperature.  Persons  who 
had  recently  recovered  from  an  attack  of  erysipelas 
proved  to  be  immune.  These  experiments  were  under- 
taken on  the  ground  that  malignant  tumors  had  previ- 
ously been  found  to  improve  or  entirely  disappear  in 
persons  who  had  recovered  from  accidental  erysipelas. 
During  the  last  few  years  this  fact  has  been  therapeu- 
tically  applied  to  the  treatment  of  malignant  tumors 
by  the  artificial  production  of  erysipelas  by  the  inocu- 
lation of  pure  cultures  of  streptococcus  or  of  their  toxic 
products,  and  in  some  cases  of  sarcomata,  with  con- 
siderable success.  In  carcinomata  the  results  have 
been  very  slight.  In  this  country  the  experimental 
work  upon  this  subject  and  the  actual  treatment  of 


STREPTOCOCCUS  PYOGENES.  485 

cases  has  been  largely  carried  out  by  or  under  the 
direction  of  Dr.  Coley.  He  has  kindly  sent  me  the 
following  notes  on  his  results  : 

"  The  improvement  and  inhibitory  action  which  the 
toxins  have  upon  carcinoma  have  proved  to  be,  in 
nearly  all  cases,  but  temporary,  and  in  no  case  has 
the  disease  remained  in  abeyance  sufficiently  long  to  be 
regarded  as  cured. 

"  On  the  other  hand,  in  sarcoma,  which  is  the  only 
form  of  malignant  tumor  in  which  I  have  advocated 
the  treatment,  sufficient  time  has  elapsed  to  enable  us 
to  draw  the  following  conclusions  : 

"  The  toxins  injected  subcutaneously  into  the  tissues, 
either  into  the  tumor  substance  or  into  parts  remote 
from  the  tumor,  exercise  a  distinctly  inhibitory  action 
upon  the  growth  of  all  varieties  of  sarcoma.  This 
action  is  the  least  marked  in  melanotic  sarcoma,  and 
tli us  far  no  cases  of  this  form  of  tumor  have  disap- 
peared under  the  treatment.  The  influence  of  the 
toxins  upon  round-celled  sarcoma  is  much  more  pow- 
erful than  it  is  upon  melanotic,  although  distinctly  less 
than  upon  the  spindle-celled  variety.  A  number  of 
cases  of  round-celled  sarcoma  in  which  the  diagnosis 
was  unquestioned  disappeared,  and  the  patients  have 
remained  well  beyond  three  years.  Nearly  half  of  the 
cases  treated  showed  no  appreciable  decrease  in  size;  the 
majority  of  the  others  which  did  show  marked  improve- 
ment at  first,  after  decreasing  in  size  for  a  few  weeks, 
again  began  to  increase  and  were  no  longer  influenced 
by  the  treatment. 

"  In  half  of  the  cases  of  spindle-celled  sarcoma 
treated  by  the  toxins  the  disease  has  disappeared 
entirely,  and  the  majority  of  the  successful  cases  have 


486  BACTERIOLOGY. 

remained  well  sufficiently  long  to  justify  their  being 
regarded  as  cured.  It  should  be  distinctly  stated  that 
all  of  the  tumors  under  consideration  were  inoper- 
able, as  I  have  never  advised  treatment  except  in  such 
cases. 

"  It  is  a  curious  fact,  from  the  stand-point  of  path- 
ology, that  the  largest  percentage  of  successful  cases 
has  occurred  in  the  spindle-celled  variety,  the  very  one 
in  which  errors  of  diagnosis  are  practically  impossible. 
In  addition  to  microscopical  examinations  by  the  best  of 
pathologists,  the  malignancy  of  the  tumors  was  further 
confirmed  by  the  characteristic  clinical  appearances, 
and  in  many  cases  by  a  history  of  repeated  recurrences. 

•'  I  have  now  three  cases  of  spindle-celled  sarcoma 
which  have  remained  well  beyond  three  years;  one  case 
of  mixed  (round  and  spindle)  celled,  which,  after  re- 
maining well  three  and  one-fourth  years,  had  a  return 
in  the  abdomen,  and  died  about  eight  months  later. 
This  case  certainly  would  establish  the  correctness  of 
the  early  diagnosis. J> 

Dr.  Coley  would  be  the  first  to  acknowledge  that 
even  the  very  moderate  claims  put  forward  in  this 
communication  are  disputed  by  many  surgeons,  they 
claiming  that  the  disappearance  of  the  tumors  is  due 
to  other  causes  than  the  treatment.  In  spite,  however, 
of  the  treatment  being  frequently  deleterious  to  the 
general  health,  and  the  occurrence  from  time  to  time 
of  the  spontaneous  disappearance  of  apparently  malig- 
nant tumors,  I  think  we  must  allow  that  the  proof  is 
very  strong  that  some  sarcomatous  tumors  have  been 
arrested  and  caused  to  disappear  by  the  toxin  injec- 
tions, and  that  where  they  are  clearly  inoperative  and 
progressing  the  treatment  should  be  tried. 


STREPTOCOCCUS  PYOGENES.  487 

Production  of  Toxic  Substances.  There  is  no  doubt 
that  this  micrococcus  causes  fever,  general  symptoms 
of  intoxication,  and  death  by  means  of  toxic  substances 
which  it  forms  in  its  growth;  but  what  these  substances 
are — whether  they, are  due  to  splitting  up  of  animal 
proteids,  or  are  secretion-products,  or  whether  they  are 
contained  in  the  cell-bodies  of  the  organism — what 
their  composition  is  and  how  they  are  produced  in  cul- 
tures we  do  not  know. 

Susceptibility  to  Streptococcus  Infection.  As  with  the 
other  ever-present  pus  cocci,  the  staphylococci,  which 
have,  as  a  rule,  only  slight  virulence,  the  streptococcus 
is  more  likely  to  invade  the  tissues,  forming  abscesses 
or  erysipelatous  and  phlegmonous  inflammation  in  man 
when  the  standard  of  health  is  reduced  from  any  cause, 
and  especially  when  by  absorption  or  retention  various 
toxic  organic  products  are  present  in  the  body  in  excess. 
It  is  thus  that  the  liability  to  these  local  infections,  as 
complications  or  sequelae  of  various  specific  infectious 
diseases,  in  the  victims  of  chronic  alcoholism,  and  con- 
stitutional affections  in  those  exposed  to  septic  emana- 
tions from  sewers,  etc.',  and  probably  in  many  cases 
from  the  absorption  of  toxic  products  formed  in  the 
alimentary  canal  as  a  result  of  the  iugestion  of  im- 
proper food,  or  of  abnormal  fermentative  changes  in 
the  contents  of  the  intestine,  or  from  constipation,  are 
to  be  explained. 

Immunity.  Knorr  succeeded  in  producing  a  moder- 
ate immunity  in  rabbits  against  an  intensely  virulent 
streptococcus  by  injections  of  very  slightly  virulent 
cultures.  Pasquale  was  able  to  immunize  these  ani- 
mals partially  against  septicaemia.  Marmorek  has 
immunized  sheep,  asses,  and  horses  against  very  large 


488  BACTERIOLOGY. 

doses  of  a  streptococcus,  which  though  but  slightly 
virulent  for  them  was  intensely  so  for  rabbits. 

In  none  of  the  streptococcus  inflammations  do  we 
notice  much  apparent  tendency  to  the  production  of 
immunizing  and  curative  substances  in  the  blood  by  a 
single  infection. 

Severe  general  infections  usually  progress  to  a  fatal 
termination  after  a  few  days,  weeks,  or  months.  It  is 
true,  however,  that  cases  of  erysipelas,  cellulitis,  and 
abscess,  after  periods  varying  from  a  few  days  to 
months,  tend  to  recover,  and  to  a  certain  extent,  there- 
fore, we  may  assume  protective  processes  have  been 
called  forth.  In  these  cases,  however,  we  know  from 
experience  that  faulty  treatment,  by  lessening  the  local 
or  general  resistance,  would,  as  a  rule,  cause  the  sub- 
siding infection  to  again  progress  and  that  to  perhaps  a 
more  serious  extent  than  the  original  attack.  Koch 
and  Petruschky  tried  a  most  interesting  experiment. 
They  inoculated  cutaneously  a  man  suffering  from  a 
malignant  tumor  with  a  streptococcus  obtained  from 
erysipelas.  He  developed  a  moderately  severe  attack, 
whi'ch  lasted  about  ten  days.  On  its  subsidence  they 
reinoculated  him;  a  new  attack  developed,  which  ran 
the  same  course  and  over  the  same  area.  This  was 
repeated  ten  times  with  the  same  results. 

This  experiment  proved  that  in  this  case,  at  least, 
little  if  any  lasting  curative  or  immunizing  substances 
were  produced  by  repeated  attacks  of  erysipelas,  and 
that  the  recovery  from  each  attack  was  due  to  local 
and  transitory  protective  developments. 

The  severe  forms  of  infection,  such  as  septicaemia 
following  injuries,  operations,  and  puerperal  infections, 
show  little  tendency  to  be  arrested  after  being  well 


STREPTOCOCCUS  PYOGENES.  489 

established.  Having,  then,  in  remembrance  the  above 
facts,  let  us  consider  the  results  already  obtained  in  the 
experimental  immunization  and  treatment  of  animals 
and  men  suffering  from  or  in  danger  of  infection  with 
streptococci.  One  method  is  now  chiefly  used  for  the 
immunization  and  attempt  to  produce  curative  sub- 
stances in  animals.  The  living,  virulent  streptococcus 
itself  is  injected  in  gradually  increasing  doses.  Mar- 
morek1  was  the  first  to  attempt  to  produce  a  curative 
serum  on  a  large  scale. 

Influence  of  Serum  from  Animals  Immunized  Against 
Streptococcus  Infection  upon  Streptococcus  Infections 
in  Other  Animals. 

The  results  reported  since  Marmorek's  commu- 
nication in  1895  upon  the  immunizing  effects  of  anti- 
streptococcic  serum  in  animals  have  been  very  vari- 
able. 

Reliable  positive  results  are,  however,  more  impor- 
tant than  negative  ones,  since  they  indicate  under 
proper  conditions  what  can  be  accomplished.  This  is 
certainly  true  if  at  the  same  time  we  can  find  good 
reasons  for  the  failures  reported. 

For  the  data  in  the  following  table  I  am  indebted  to 
Anna  W.Williams,  assistant  bacteriologist  in  the  Health 
Department  Laboratories.  For  the  use  of  the  same  I 
wish  to  express  my  appreciation, 

In  this  present  table  are  given  the  results  following 
the  injection  of  small  amounts  of  a  serum  which  repre- 
sents in  immunizing  value  what  about  one-third  of  the 
horses  are  able  to  produce.  In  the  following  experi- 

i  Annales  de  1'Institut  Pasteur,  July,  1895. 


490 


BACTERIOLOGY. 


ments  the  serum  and  culture  were  injected  subcutane- 
ously  in  rabbits  at  the  same  time,  but  in  opposite  sides 
of  the  body  : 

TABLE  SHOWING  STRENGTH  OF  AVERAGE  GRADE  OF  ANTI- 

STREPTOCOCCIC  SERUM  GIVEN  BY  SELECTED  HORSES  AFTER 

SIX  MONTHS  OF  INJECTION   OF    SUITABLE    AMOUNTS  OF 
LIVING  STREPTOCOCCI. 


Weight 
of 
rabbit. 

Amounts 
inoculated. 

Result. 

Autopsy. 

Serum  and  culture  : 

Grms. 

Serum.     Cult. 

1.  Inoculated  at  same  time 

1430 

0.25  c.c.  0.01  c.c. 

Lived 

2. 

1350 

0.125"    0.01    " 

" 

3. 

1600 

0.25    "    0.01    " 

" 

4.  Subcutaneously      " 

1440 

0.25    "    0.01    " 

" 

5.  On  opposite  sides   " 

1770 

0.1      "    001    " 

" 

6.    '•       "            "       " 

1630 

0.1      "    0.01    " 

" 

Controls  : 

1.  Rabbits   injected  with 
culture  only 
2. 

3 

1750 

1870 
1820 

0.001  " 
0.001  " 
0.01    " 

Died  in 
4  days. 
Died  in 
24hrs. 
Died  in 
4  days. 

Strept.  in- 
fection. 

The  above  results  have  been  repeatedly  obtained,  and 
are  absolutely  conclusive  that,  as  Marmorek  and  others 
have  claimed,  the  serum  of  properly  selected  animals, 
which  have  been  repeatedly  injected  with  living  strep- 
tococci in  suitable  doses,  possesses  bactericidal  properties 
upon  the  same  streptococcus  when  it  comes  in  contact 
with  it  within  the  bodies  of  animals. 

Definite  protection  from  the  serum  has  been  obtained 
by  many  reliable  observers  since  Marmorek' s  first 
reports. 


STREPTOCOCCUS  PYOGENES.  491 

Is  Protection  Afforded  by  the  Same  Serum  Against  All 
Varieties  of  Streptococci? 

We  have  tested  the  protective  value  of  one  serum 
against  five  streptococci.  First,  the  streptococcus  given 
us  by  Marmorek,  which  was  obtained  from  a  case  of 
angina  complicating  scarlet  fever.  Its  virulence  is  now 
such,  after  having  passed  through  hundreds  of  rabbits, 
that  0.000001  c.c.  is  the  average  fatal  dose.  Second, 
a  streptococcus  obtained  from  a  case  of  erysipelas  in 
England.  Its  virulence  is  0.00001  c.c.  on  the  average. 
Third,  a  streptococcus  obtained  from  a  case  of  cellu- 
litis  a  few  weeks  ago,  its  virulence  being  about  6  c.c. 
Fourth,  a  streptococcus  sent  me  by  Theobald  Smith. 
Its  virulence  is  such  that  0.1  c.c.  is  the  average  fatal 
dose.  Fifth,  another  culture  sent  me  by  Smith,  which 
grew  in  short  chains  and  was  obtained  from  milk;  its 
virulence  was  similar  to  No.  4. 

Against  the  first  three  streptococci  derived  from  three 
different  varieties  of  infection  existing  in  three  different 
countries  the  serum  produced  in  the  horse  by  the  strep- 
tococcus from  England  had  nearly  the  same  value. 
Against  the  latter  two  streptococci,  as  well  as  against 
a  pneumococcus,  which  in  ordinary  cultures  looks  like 
a  streptococcus,  the  serum  had  no  effect. 

The  results  published  by  others  must  also  be  taken 
to  prove  that  a  serum  which  protects  from  infection 
with  one  streptococcus  may  fail  against  others;  but, 
taking  all  together,  they  indicate  that  the  majority  of 
streptococci  met  with  in  practice  will  be  influenced  by 
the  same  serum.  Many  more  streptococci,  however, 
must  be  obtained  from  human  infections  and  tested 
before  we  can  be  certain  of  this.  Those  obtained  from 


492  BACTERIOLOGY. 

human  sepsis,  which  are  not  very  virulent  in  animals, 
are  especially  in  need  of  investigation.  If  those  who 
use  the  serum  will  send  to  the  laboratories  materials 
for  cultures  this  can  in  time  be  fully  determined. 

The  Preparation  of  the  Serum.  Antistreptococcus 
serum  is  obtained  from  the  horse,  ass,  and  sheep  after 
treatment  by  repeated  injections  of  living  streptococcus 
cultures.  The  procuring  of  a  serum  of  the  highest 
potency  requires  a  considerable  number  of  animals,  for 
some  produce  with  the  same  treatment  a  more  protec- 
tive serum  than  others.  The  serum  must  be  sterile 
from  streptococcus  as  well  as  from  other  contaminations. 

The  Stability  of  the  Serum.  Unfortunately,  after  sev- 
eral weeks  or  months,  the  serum,  as  a  rule,  at  least,  loses 
its  protective  value.  It  should  be  kept  in  a  cold  and 
dark  place.  Not  only  ourselves,  but  others,  such  as 
Aronson,  have  found  this  to  be  true. 

To  this  deterioration  can  probably  be  ascribed  the 
failure  of  Koch,  Petruschky  and  others  to  find  in  the 
serum  any  power  to  protect  animals  from  infection. 

The  Standardization  of  the  Value  of  the  Serum.  The 
value  of  the  serum  is  measured  by  the  amount  required 
to  protect  against  a  multiple  of  a  fatal  dose  of  a  very 
virulent  streptococcus.  The  dose  is  usually  a  thousand 
times  the  average  fatal  amount  of  a  very  virulent 
streptococcus. 

This  method  gives,  as  a  rule,  to  those  unfamiliar 
with  bacteriology  an  exaggerated  idea  of  the  potency 
of  the  serum. 

A  thousand  times  the  amount  of  a  very  virulent 
streptococcus  culture  required  to  kill  an  animal  by 
producing  septicaemia  is  still  too  little  to  kill  by  the 
streptococci  injected;  it  is  only  their  enormous  multi- 
plication in  the  animal  which  kills. 


STREPTOCOCCUS  PYOGENES.  493 

Double  the  fatal  dose  of  a  culture  which  kills  only 
in  a  dose  of  10  c.c.  or  over  is  a  more  severe  test  than 
a  thousand  times  a  very  virulent  one. 

It  is  entirely  different  in  case  of  an  antitoxin 
which  does  not  prevent  primarily  the  growth  of  the 
germ,  but  neutralizes  a  chemical  substance — its  toxin. 

Its  Therapeutic  Results.  To  estimate  the  exact  pre- 
sent and  future  value  of  antistreptococcus  serum  is  a 
matter  of  the  utmost  difficulty.  Many  of  the  cases 
reported  are  of  little  or  no  help,  because,  on  account  of 
no  cultures  having  been  made,  we  are  in  doubt  as  to  the 
nature  of  the  bacterial  infection.  Even  when  bacterio- 
logical examinations  are  made  during  life  in  cases  of 
septicsemia,  they  are  apt  to  fail  to  give  us  any  informa- 
tion. Under  Marmorek's  supervision  many  cases  have 
been  injected;  thus,  even  as  far  back  as  June,  1895, 
when  his  last  statistics  were  published,  he  had  treated 
96  cases  of  scarlet  fever,  411  cases  of  erysipelas,  16 
cases  of  puerperal  fever,  and  smaller  numbers  of  cases 
of  tonsillitis  and  of  post-operative  septicsemia. 

Since  then  he  has  treated  many  forms  of  phthisis. 
In  all  these  cases  marked  improvement  is  reported  to 
have  followed  when  they  were  due  to  streptococci. 
Thus,  in  sixteen  cases  of  puerperal  fever  seven  were 
due  to  streptococcus  alone.  All  these  recovered. 
Three  were  due  to  the  streptococcus  and  colon  bacil- 
lus and  one  to  the  colon  bacillus  alone.  These  four 
all  died.  In  five,  streptococci  were  associated  with 
staphylococci.  Two  of  these  died,  three  recovered. 

In  phthisis  where  no  cavities  have  as  yet  appeared 
the  fever  and  sweats  lessened  and  all  symptoms  im- 
proved. He  did  not  state  that  any  cases  were  abso- 
lutely cured.  Marmorek's  results  are  by  far  the  best 
reported,  and  without  casting  any  doubt  upon  the  in- 


494  BACTERIOLOGY. 

tended  honesty  of  his  conclusions,  it  is  my  conviction 
that  they  give  undoubtedly  too  favorable  a  view  of  the 
value  of  the  serum. 

In  the  comparatively  few  cases  of  puerperal  fever, 
wound  infection,  scarlet  fever,  and  bronchopneumonia 
that  we  have  seen  under  the  treatment  the  apparent  re- 
sults have  not  been  uniform.  Only  occasionally  did 
we  see  results  which  appeared  to  be  distinctly  due  to 
the  serum. 

In  a  number  of  cases  of  septicaemia  where  chills  had 
occurred  daily  for  days  they  ceased  absolutely  or 
lessened  under  daily  doses  of  20  to  50  c.c.  The 
temperature,  though  ceasing  to  rise  to  such  high  eleva- 
tions, did  not  average  more  than  one  or  two  degrees 
lower  than  before  the  injections.  The  serum  treat- 
ment was  kept  up  for  four  weeks.  Some  cases  conva- 
lesced; others  after  a  week  or  more  grew  worse  and  died. 

In  some  cases  the  temperature  fell  immediately  upon 
giving  the  first  injection  of  serum,  and  after  subse- 
quent injections  remained  normal,  and  the  cases  seemed 
greatly  benefited.  As  a  rule,  in  these  cases  no  strep- 
tococci or  any  other  organisms  were  obtained  from  the 
blood.  On  bronchopneumonia,  laryngeal  diphtheria, 
and  in  phthisis  we  have  seen  absolutely  no  effect. 

The  results  obtained  here  in  New  York  by  both 
physicians  and  surgeons  have  not,  on  the  whole,  been 
very  encouraging. 

In  some  of  the  cases  where  apparently  favorable  re- 
sults were  obtained  other  bacteria  than  streptococci  were 
found  to  be  the  cause  of  the  disease.  We  believe  that 
the  following  conclusions  will  be  found  fairly  accurate  : 

A  single  antistreptococcic  serum  protects  healthy 
rabbits  from  infection  from  most  of  the  streptococci 
obtained  from  human  sepsis  due  to  the  streptococcus, 


STREPTOCOCCUS  PYOQENES.  495 

but  not  from  all.  Failure  to  do  good  in  human  infec- 
tion cannot,  as  a  rule,  be  attributed  to  the  variety  of 
streptococcus.  The  serum  will  in  animals  limit  an  in- 
fection already  started  if  it  has  not  progressed  too  far. 
The  apparent  therapeutic  results  in  cases  of  human 
streptococcus  infection  are  variable.  In  some  cases 
the  disease  has  undoubtedly  advanced  in  spite  of  large 
injections,  and  here  it  has  not  seemed  to  have  had 
any  effect.  In  other  cases  good  observers  rightly  or 
wrongly  believe  they  have  noticed  great  improvement 
from  it.  Except  rashes,  few  have  noticed  deleterious 
results,  although  very  large  doses  have  been  followed 
in  several  instances,  for  a  short  time,  by  albuminous 
urine  and  even  temporary  suppression. 

In  suitable  cases  we  are,  I  think,  warranted  in  try- 
ing it,  but  we  must  not  expect  very  striking  results. 

For  our  own  satisfaction,  and  to  increase  our  knowl- 
edge, we  should  always  have  satisfactory  cultures  made 
when  possible,  and  the  streptococci,  if  obtained,  tested 
with  the  serum  used  in  the  treatment.  In  the  cases 
where  we  want  most  to  use  the  serum,  such  as  puerperal 
fever,  septicaemia,  ulcerative  endocarditis,  etc.,  we 
find  that  it  is  very  difficult  to  make  a  bacteriological 
diagnosis  from  the  symptoms,  and  in  over  one-half  of 
the  cases  even  the  bacteriological  examination  carried 
out  in  the  most  thorough  way  will  fail  to  detect  the 
special  variety  of  bacteria  causing  the  infection.  This 
is  often  a  great  hinderance  to  the  proper  use  of  curative 
antistreptococcic  serum,  for  it,  of  course,  has  no  specific 
effect  upon  the  course  of  any  infection  except  that  due 
to  the  streptococcus. 

Care  should  be  taken  to  get  only  recently  tested 
serum,  for  after  six  weeks  the  best  serum  is  almost 
inert;  much  on  the  market  is  worthless,  and  as  it  is 


496  BACTERIOLOGY. 

weak,  and  the  testing  for  strength  is  still  very  crude, 
full  doses  of  serum  should  be  given  if  the  case  is  at  all 
serious,  for  the  dose  is  limited  only  by  the  amount 
of  horse-serum  which  we  feel  it  safe  to  give,  not  be- 
cause we  have  sufficient  protective  substance. 

Bacteriological  Diagnosis.  Streptococci,  using  the 
name  in  its  broadest  sense,  can  often  be  demonstrated 
microscopically  by  simply  making  a  smear  preparation 
of  the  suspected  material  and  staining  with  methylene- 
blue  solution  or  diluted  ZiehPs  fluid.  In  order  to 
demonstrate  them  microscopically  in  the  tissues,  the 
sections  are  best  stained  by  Kuhne's  methylene-blue 
method.  In  all  cases,  even  when  the  microscopical 
examination  fails,  the  cocci  may  be  found  by  the  culture 
method  on  plate  agar  or  slanted  agar  at  37°  C.  To 
obtain  them  from  a  case  of  erysipelas  it  is  best,  accord- 
ing to  Fehleisen,  to  excise  a  small  piece  of  skin  from 
the  margin  of  the  erysipelatous  area  in  which  the  cocci 
are  most  numerous;  this  is  crushed  up  and  part  of  it 
transfered  to  a  gelatin  tube  and  to  the  melted  agar  in 
another  tube.  After  shaking  thoroughly  the  contents 
are  poured  out  into  Petri  dishes.  The  gelatin  is  kept 
at  a  temperature  of  20°  C.  At  the  end  of  two  or 
three  days  numerous  small  colonies  develop  in  the 
vicinity  of  the  particles  of  skin.  The  agar  plate  is 
kept  at  37°  C.  for  twenty-four  hours.  It  is  usually 
sufficient,  however,  to  make  a  streak  culture  on  agar  in 
a  Petri  dish  with  the  crushed  excised  portion  of  skin 
and  place  this  in  the  incubator  at  37°  C. 

In  septicaemia  the  culture  method  is  always  required 
to  demonstrate  the  presence  of  streptococci,  as  the  micro- 
scopical examination  of  specimens  of  blood  is  not  suffi- 
cient. For  this  purpose  from  3  to  5  c.c.  of  the  blood 
should  be  drawn  from  the  vein  of  the  arm  aseptically 


STREPTOCOCCUS  PYOGENES.  497 

by  means  of  a  hypodermatic  needle,  and  each  c.c.  added 
to  a  tube  of  broth,  in  order  to  produce  an  adequate 
development  of  the  cocci,  which  are  found  in  small 
numbers  in  the  bloodvessels.  Petruschky  is  of  the 
opinion  that  the  cocci  can  best  be  shown  in  blood  by 
animal  inoculation.  Having  withdrawn  from  the 
patient  10  c.c.  of  blood  by  means  of  a  hypodermatic 
syringe,  under  aseptic  precautions,  he  injects  a  portion 
of  this  into  the  abdominal  cavity  of  a  mouse,  while  the 
other  portion  is  planted  in  bouillon.  Mice  thus  inocu- 
lated die  from  septicaemia  when  virulent  streptococci 
are  present  only  in  very  small  numbers  in  the  blood. 
If  a  successful  inoculation  takes  place  we  can,  through 
the  absence  or  presence  of  the  development  of  capsules, 
often  differentiate  between  the  pneumococcus  and  the 
streptococcus,  which  cultures  may  fail  to  do.  The 
morphological  and  cultural  characteristics  of  the  strep- 
tococcus give  us,  unfortunately,  no  absolute  knowledge 
as  to  the  influence  which  the  protecting  serum  will 
have.  The  actual  test  is  here  our  only  method.  The 
detection  of  the  streptococcus  in  the  blood  is  in  itself 
an  unfavorable  prognostic  sign. 

The  blood  cultures  in  perhaps  the  majority  of  cases 
of  septicaemia  give  no  positive  results,  for  many  of 
these  cases  develop  their  symptoms  and  even  die  from 
the  absorption  of  toxins  from  the  local  infection,  such 
as  an  amputation  wound  or  an  infected  uterus  or  peri- 
toneum, and  the  bacteria  never  invade  the  blood.  When 
we  get  negative  results  we  are,  as  a  rule,  utterly  unable 
to  test  the  case  with  curative  serums  with  any  accuracy, 
for  the  sepsis  may  be  due  to  either  the  streptococcus, 
colon  bacillus,  staphylococcus,  or  a  number  of  other 
pathogenic  varieties  of  bacteria. 

32 


CHAPTER  XXVIII. 

MICROCOCCUS  LANCEOLATUS  (PNEUMOCOCCUS  ;  MICRO- 
COCCUS  PNEUMONIA  CROUPOS2E  OF  STERNBERG; 
MICROCOCCUS  OF  SPUTUM  SEPTIC^MIA  AND  DIPLO- 
COCCUS  OF  FRAENKEL;  DIPLOCOCCUS  PNEUMONIA 
OF  WEICHSELBAUM). 

THIS  micrococcus  was  first  observed  by  Sternberg,  in 
1880,  in  the  blood  of  rabbits  inoculated  with  his  own 
saliva  (and  almost  simultaneously  by  Pasteur  under 
similar  conditions),  whence  it  was  called  by  Sternberg 
micrococcus  Pasteuri.  It  was  subsequently  described 
by  Talamon  (1883),  and  demonstrated  by  him  to  be 
capable  of  producing  fibrinous  pneumonia  in  rabbits 
when  introduced  into  the  parenchyma  of  the  lung  of 
these  animals.  In  1885  and  1886  this  micro-organism 
was  subjected  to  an  extended  series  of  investigations 
by  A.  Fraenkel,  Sternberg,  Weichselbaum,  Netter 
and  others,  and  proved  by  them  to  be  the  chief  etio- 
logical  factor  in  the  production  of  lobar  or  croupous 
pneumonia  in  man. 

Morphology.  Very  irregular;  occurs  as  spherical  or 
oval  cocci,  usually  united  in  pairs,  but  sometimes  in 
longer  or  shorter  chains  consisting  of  from  three  to  six 
or  more  elements  and  resembling  the  streptococcus.  The 
individual  cells,  as  they  commonly  occur  in  pairs,  are 
somewhat  oval  in  shape,  being  usually  pointed  at  one 
end — hence  the  name  lanceolatus,  or  lancet-shaped. 


MICROCOCCUS  LANCEOLATUS. 


499 


When  thus  united  the  junction,  as  a  rule,  is  between 
the  broad   ends  of   the  oval,  with  the  pointed  ends 


FIG.  65. 


Diplococcus  of  pneumonia  from  blood,  with  surrounding  capsule. 
FIG.  66. 


Pneumococcus  from  bouillon  culture,  resembling  streptococcus. 

turned  outward;  but  variation  in  form  and  arrange- 
ment of  the  cells  is  characteristic  of  this  organism, 
there  being  great  differences  according  to  the  source 


500  BACTERIOLOGY. 

from  which  they  are  obtained.  As  observed  in  the 
blood  of  inoculated  animals  it  is  usually  in  pairs  of 
lancet-shaped  elements,  which  are  surrounded  by  a  cap- 
sule. (See  Fig.  65.)  When  grown  on  culture  media 
longer  or  shorter  chains  are  frequently  formed,  which 
can  scarcely  be,  or  even  not  at  all,  distinguished  from 
chains  of  streptococci.  The  individual  cells  are  almost 
spherical  in  shape,  and  they  are  rarely  surrounded  by 
a  capsule.  (See  Fig.  66.) 

The  capsule  is  best  seen  in  stained  preparations  from 
the  blood  and  exudates  of  fibrinous  pneumonia  or  from 
the  blood  of  an  inoculated  animal,  especially  the  mouse, 
in  which  it  is  commonly,  though  not  always,  present. 
It  is  seldom  seen  in  preparations  from  cultures. 

It  stains  readily  with  ordinary  aniline  colors;  it  is 
not  decolorized  after  staining  by  Gram's  method.  The 
capsule  may  be  demonstrated  in  blood  or  sputum  either 
by  Gram's  or  Welch's  (glacial  acetic  acid)  method. 

Biological  Characters.  It  grows  on  almost  all  the 
culture  media  ordinarily  employed,  but  its  suscepti- 
bility is  shown  not  only  by  its  irregularity  of  form,  but 
also  by  its  slow  and  comparatively  scanty  growth  and 
by  its  rapid  loss  of  virulence  and  power  of  reproduction 
under  varying  conditions.  It  grows  equally  well  in 
the  absence  as  in  the  presence  of  oxygen,  being  thus 
both  aerobic  and  facultative  anaerobic;  its  parasitic 
nature  is  exhibited  by  the  short  range  of  temperature 
at  which  it  grows — viz.,  from  25°  to  42°  C. — its  maxi- 
mum growth  being  at  about  37°  C.,  or  the  temperature 
of  the  body.  Its  thermal  death-point,  as  determined 
by  Sternberg,  is  52°  C.,  the  time  of  exposure  being 
ten  minutes.  It  loses  its  vitality  in  cultures  in  a 
comparatively  short  time,  and  is  very  sensitive  to  the 


MICEOCOCCUS  LANCEOLATUS.  501 

action  of  germicidal  agents.  Its  pathogenic  power  also 
undergoes  attenuation  very  rapidly  when  cultivated  on 
artificial  media.  It  is  non-motile. 

In  the  cultivation  of  this  organism  one  of  the  most 
important  considerations  is  the  reaction  of  the  media 
employed.  According  to  Fraenkel,  Sternberg  and 
others,  it  grows  only  in  culture  media  when  they  have 
a  slightly  alkaline  reaction.  Kruse  and  Pansini  showed 
by  their  investigations  that,  according  to  the  source 
from  which  it  was  obtained,  it  grows  at  times  equally 
well  in  a  slightly  alkaline  or  slightly  acid  medium. 
Not  infrequently,  however,  all  experimenters  have 
found  that  no  growth  at  all  occurs,  irrespective  of  the 
composition  or  reaction  of  the  media  employed.  The 
weight  of  opinion,  nevertheless,  seems  to  be  in  favor  of 
the  selection  of  a  slightly  alkaline  medium. 

The  organism  grows,  as  has  been  said,  on  all  the  cul- 
ture media  ordinarily  employed  for  the  cultivation  of 
bacteria — viz.,  on  agar  and  gelatin,  in  bouillon,  ascitic 
fluid,  and  blood-serum.  The  best  medium  for  its 
growth  is  a  mixture  of  one-third  ascitic  or  pleuritic 
fluid  and  two-thirds  bouillon.  It  grows  readily  in 
milk,  causing  coagulation  with  the  production  of  acid, 
though  this  is  not  constant. 

Growth  on  Agar.  Cultivated  on  plain  nutrient  agar, 
after  forty-eight  hours  in  the  incubator,  there  ap- 
pears a  thin,  colorless  layer  composed  of  dilute  non- 
confluent  colonies.  If  blood-serum  or  ascitic  fluid  be 
added  to  the  agar  the  individual  colonies  are  larger  and 
closer  together,  and  the  growth  is  more  distinct  in  con- 
sequence and  of  a  grayish  color.  The  surface  colonies 
resemble  those  of  some  of  the  streptococci  growing  in 
short  chains;  they  are  almost  circular  in  shape,  finely 


502  BACTERIOLOGY. 

granular  in  structure,  and  have  a  somewhat  darker, 
more  compact  centre,  surrounded  by  a  paler  marginal 
zone.  With  high  magnification  rows  of  cocci  are  seen 
sprouting  from  the  edges.  In  stick  cultures  along  the 
line  of  puncture  minute  transparent  drops  appear. 

Growth  on  Gelatin.  The  growth  on  gelatin  is  slow, 
if  there  is  any  development  at  all,  owing  probably  to 
the  low  temperature— viz.,  22°  to  24°  C.— at  which  the 
gelatin  has  to  be  kept.  The  gelatin  is  not  liquefied. 

Growth  on  Blood-serum,  The  growth  on  Ldffler's 
blood-serum  mixture  is  very  similar  to  that  on  agar, 
but  somewhat  more  vigorous,  appearing  on  the  surface 
as  a  delicate  layer  of  dew-like  drops. 

Growth  in  Bouillon.  In  bouillon,  at  the  end  of  twelve 
to  twenty-four  hours  in  the  incubator,  a  slight  cloudi- 
ness of  the  liquid  will  be  found  to  have  been  pro- 
duced, due  to  the  development  of  the  micrococci, 
which  on  microscopical  examination  can  be  seen  to  be 
arranged  in  pairs  or  longer  and  shorter  chains.  After 
two  or  three  days  the  medium  becomes  again  trans- 
parent, owing  to  the  subsidence  of  the  cocci  to  the 
bottom  of  the  tube. 

Special  Media.  Fraenkel  was  the  first  to  draw  at- 
tention to  the  fact  that  this  organism  soon  loses  its 
reproductive  power  when  grown  on  ordinary  culture 
media,  and  more  particularly  solid  media.  In  fluid 
media  the  vitality  is  not  quite  so  quickly  lost;  but 
even  here  it  is  found  advisable  in  practice  to  transplant 
fresh  cultures  every  day.  By  this  method,  when  bou- 
illon cultures  are  used,  the  vitality  may  be  indefinitely 
prolonged;  but  after  transplantation  through  several 
generations  it  is  found  that  the  cultures  begin  to  lose 
in  virulence,  which  finally  disappears  entirely.  In  order 


MICROCOCCUS  LANCEOLATUS.  503 

to  restore  this  virulence,  or  to  keep  it  from  becoming 
attenuated,  it  is  necessary,  therefore,  to  interrupt  the 
transplantation  and  pass  the  organism  through  the 
bodies  of  susceptible  animals. 

With  the  view  of  overcoming  some  of  these  obstacles 
in  the  way  of  cultivating  this  micrococcus,  several  spe- 
cial media  have  been  proposed  by  various  experimenters 
in  the  place  of  the  ordinary  culture  media.  The  best 
fluid  medium  for  the  growth  of  the  pneumococcus  is 
Marmorek's  mixture,  consisting  of  bouillon,  2  parts  ; 
ascitic  or  pleuritic  fluid,  1  part.  In  this  fluid  pneumo- 
cocci  grow  well,  and  cultures  when  preserved  in  a  cool 
place  and  prevented  from  drying  retain  their  vitality 
and  also  their  virulence  for  a  number  of  months. 
Lambert  has  found  cultures  in  this  medium  alive  and 
fully  virulent  after  eight  months. 

Loftier7  s  blood-serum  mixture  is  probably  the  best 
solid  tube  medium  for  making  cultures,  and  is  very 
convenient  and  useful  at  autopsies.  One  and  one-half 
per  cent,  fluid  nutrient  agar  mixed  with  one-third  its 
quantity  of  warm  ascitic  fluid  makes  an  excellent  plate 
medium. 

Effects  of  Germicidal  Agents,  Light  and  Drying.  The 
following  are  the  effects  of  germicidal  and  antiseptic 
agents  on  this  organism,  according  to  observations 
made  by  Sternberg  :  Boric  add,  saturated  solution, 
failed  to  destroy  the  vitality  after  two  hours,  but  a  so- 
lution of  1  :  400  restrained  its  development;  carbolic 
acid,  1  per  cent,  solution,  destroys  the  vitality  in  two 
hours,  and  1 :  500  restrains  development;  mercuric  chlo- 
ride,! :  20,000,  destroys  vitality  in  two  hours,!  :  40,000 
restrains  development;  salicylic  acid  and  sodium  bibo- 
rate,  1  : 400  solution,  restrained  development. 


504  BACTERIOLOGY. 

As  to  its  duration  of  life  outside  the  body,  the 
researches  of  Bordoni-Uffreduzzi  throw  some  light. 
He  found  that  pneumonic  sputum  attached  to  clothes, 
when  dried  in  the  air  and  exposed  to  diffuse  day- 
light, retained  its  virulence,  as  shown  by  injection 
in  rabbits,  for  a  period  of  nineteen  to  fifty-five  days. 
Exposed  to  direct  sunlight  the  same  material  retained 
its  virulence  after  twelve  hours'  exposure.  This  reten- 
tion of  virulence  for  so  long  a  time  under  these  circum- 
stances is  accounted  for  by  the  protective  influence 
afforded  by  the  dried  albuminous  material  in  which  the 
micrococci  were  embedded.  Thus,  Guarnieri  observed 
that  the  blood  of  inoculated  animals,  wheu  rapidly  dried 
in  a  desiccator,  retained  its  virulence  for  months;  and 
Foa  found  that  fresh  rabbit  blood,  after  inoculation  and 
cultivation  in  the  incubator  for  twenty-four  hours, 
when  removed  at  once  to  a  cool,  dark  place,  retained 
its  virulence  for  sixty  days.  There  are  many  condi- 
tions, therefore,  in  which  the  virulence  of  the  micro- 
coccus  is  retained  for  a  considerable  length  of  time. 

The  Source  of  Infection.  Although,  as  we  have  just 
seen,  the  pneumococcus  may  retain  its  virulence  in 
dried  sputa  for  considerable  lengths  of  time,  still  such 
pneumococci  are  not  the  only  source  of  contagion,  for 
in  the  throat  secretions  of  many  healthy  persons,  and 
in  the  bronchial  and  lung  discharges  of  nearly  all  cases 
of  chronic  pulmonary  diseases,  we  have  the  pneumo- 
cocci abundantly  present. 

Pathogenesis.  The  micrococcus  lanceolatus  is  quite 
pathogenic  for  some  animals — viz.,  mice  and  rabbits — 
less  so  for  others.  In  mice  and  rabbits  the  subcutaneous 
injection  of  small  quantities  of  pneumonic  sputum  in 
the  early  stages  of  the  disease,  or  of  a  pure,  virulent 


MICROCOCCUS  LANCEOLATUS.  505 

culture  of  the  micrococcus,  usually  results  in  the  death 
of  these  animals  in  from  twenty-four  to  forty-eight 
hours.  The  course  of  the  disease  produced  and  the 
post-mortem  appearances  indicate  that  it  is  a  form  of 
septicaemia — what  is  known  as  sputum  septicaemia. 
After  injection  there  is  loss  of  appetite  and  great 
debility,  and  the  animal  usually  dies  some  time  during 
the  second  day  after  inoculation.  The  post-mortem 
examination  shows  a  local  reaction,  which  may  be  of 
a  serous,  fibrinous,  hemorrhagic,  necrotic,  or  purulent 
character;  or  there  may  be  combinations  of  all  of  these 
conditions.  The  most  marked  pathological  lesion  is 
the  enlargement  of  the  spleen,  which  in  mice  is  con- 
spicuous and  common,  and  in  rabbits  not  so  much  so. 
It  is  sometimes  hard,  dark  colored,  and  dry,  or  it  may 
be  soft  and  bright  red.  The  liver  also  is  sometimes 
dark  colored  and  gorged  with  blood,  but  more  fre- 
quently it  is  paler  than  normal  and  rich  in  fat.  The  blood 
of  inoculated  animals  immediately  after  death  often 
contains  the  micrococci  in  very  large  numbers.  For 
microscopical  examination  they  may  be  obtained  from 
the  blood  of  the  veins,  arteries,  or  cavities  of  the  heart, 
and  usually  from  the  pleural  and  peritoneal  exudations 
when  they  are  present. 

Mice  and  rabbits  are  the  most  susceptible  animals, 
and  are  thus  usually  employed  for  experimental  pur- 
poses in  investigations  with  this  micrococcus;  but 
guinea-pigs,  dogs,  cats,  rats,  and  sheep  are  also  sus- 
ceptible. Chickens  and  pigeons  are  insusceptible- 
Young  animals,  as  a  rule,  are  more  easily  affected 
than  old  ones.  In  dogs  subcutaneous  injections  usually 
give  negative  results.  True  localized  pneumonia  does 
not  usually  result  from  subcutaneous  injections  into 


506  BACTERIOLOGY 

susceptible  animals,  but  injections  made  through  the 
thoracic  walls  into  the  substance  of  the  lung  may 
induce  a  typical  fibrous  pneumonia.  This  was  first 
demonstrated  by  Talamon,  who  injected  the  fibrinous 
exudate  of  croupous  pneumonia,  obtained  after  death 
or  drawn  during  life  from  the  hepatized  portions  of 
the  lung,  into  the  lungs  of  rabbits. 

Attenuation  of  Virulence.  The  pathological  changes 
above  mentioned  apply  only  to  the  effects  produced 
by  fully  virulent  cultures  on  susceptible  animals.  With 
attenuation  of  virulence  in  the  cultures  or  decrease 
of  susceptibility  in  the  animals  different  effects  are 
produced.  When  the  disease  takes  a  rapid  course  the 
local  reaction  and  the  changes  in  the  internal  organs 
are  comparatively  slight;  but  the  longer  the  process 
lasts  the  greater  will  be  the  local  reaction  and  patho- 
logical lesions  in  the  body.  Attenuation  of  virulence 
may  be  produced  in  various  ways.  The  loss  of  viru- 
lence which  occurs  when  the  micrococcus  is  trans- 
planted in  cultures  through  several  generations  has 
already  been  referred  to.  A  similar  attenuation  of 
virulence  takes  place  also  spontaneously  in  the  course 
of  pneumonia.  Patella  has  shown  by  daily  puncture 
of  the  lung  in  different  stages  of  the  pneumonic  pro- 
cess that  the  virulence  of  the  organism  diminished  as 
the  disease  progressed,  and  that  the  nearer  the  crisis 
was  approached  the  more  attenuated  it  became — a  fact 
which  has  been  confirmed  by  others.  Welch  found 
that  the  most  virulent  micrococci  were  contained  in 
the  freshly  hepatized  portions  of  the  lung.  Fraenkel 
and  Weichselbaum  showed  that  the  cocci  taken  from 
the  lung  varied  in  virulence  according  to  the  stage  of 
the  disease  when  they  were  obtained.  Attenuation  of 


MICROCOCCUS  LANCEOLATUS.  507 

virulence  may  also  be  effected  artificially.  Banti  found 
that  the  continued  passage  through  the  bodies  of  guinea- 
pigs,  which  are  not  particularly  susceptible,  also  re- 
sulted in  a  loss  of  virulence.  Sanarelli  states  that  the 
cultivation  in  human  saliva  is  also  attended  with  an 
attenuation  of  virulence.  Cultivation  in  other  unfavor- 
able media,  or  media  to  which  substances  have  been 
added  which  restrain  development,  has  similar  attenu- 
ating effect  on  the  virulence. 

Restoration  and  Increase  of  Virulence.  The  simplest 
and  perhaps  the  most  reliable  method  of  restoring  lost 
virulence  for  any  animal  is  by  passage  through  the 
bodies  of  highly  susceptible  animals  of  the  same  species. 

Occurrence  in  Man.  The  micrococcus  lanceolatus  is 
not  infrequently  present  in  the  saliva  of  healthy  in- 
dividuals, having  been  found  by  Sternberg  in  the  oral 
cavity  of  about  20  per  cent,  of  healthy  persons  exam- 
ined. It  is  constantly  to  be  detected  in  the  rusty  sputum 
of  patients  suffering  from  acute  fibrinous  pneumonia. 
Weichselbaum  reports  having  found  it  in  94  out  of  129 
cases  of  pneumonia  examined  by  him;  Wolff  found  it 
in  65  cases  out  of  70  examined;  Netter  in  75  per  cent, 
of  his  cases,  and  in  the  sputum  of  convalescents  from 
pneumonia  in  60  per  cent.  The  more  recent  the  infec- 
tion the  greater  is  the  number  of  bacteria  found  in  the 
diseased  lung  areas.  As  the  disease  progresses  they 
decrease  in  number  until  finally  at  the  crisis  they  dis- 
appear from  the  tissues,  though  at  this  time  and  long 
after  convalescence  they  may  be  present  in  the  sputum. 
In  atypical  forms  of  pneumonia  they  may  remain  longer 
in  the  tissues,  and  in  walking  pneumonia  they  may  be 
absent  in  the  original  centres  of  infection  or  present  only 
as  attenuated  varieties,  while  the  surrounding,  newly- 


508  BACTERIOLOGY. 

formed  foci  may  contain  fully  virulent  cocci.  But 
lobar  pneumonia  is  not  the  only  form  of  pneumonia 
in  the  production  of  which  this  organism  is  concerned. 
It  has  been  shown  by  Netter  that  more  than  one-half 
of  the  cases  of  bronchopneumonia,  whether  primary  or 
secondary  to  some  other  disease,  as  measles  and  diph- 
theria, both  in  children  and  adults,  are  due  to  the 
micrococcus  lanceolatus.  The  microscopical  appear- 
ances in  bronchopneumonia  are  the  same  as  in  lobar 
pneumonia,  the  only  difference  being,  according  to  the 
observations  of  Ribbert  and  Baumgarten,  that  in  the 
former  the  infective  process  is  less  extended,  resulting 
in  the  formation  of  a  number  of  small  foci  instead  of 
the  lung  being  attacked  in  toto. 

Beside  the  affections  above  mentioned,  this  micro- 
coccus  is  associated  with  other  pathogenic  bacteria, 
producing  a  secondary  or  mixed  infection.  In  tuber- 
culosis, for  example,  it  is  often  found  associated  with 
the  tubercle  bacillus,  taking  part  with  this  organism  in 
the  destruction  of  the  tubercular  tissue  of  the  lungs. 
Having  once  reached  the  lung  it  may  penetrate  to  dif- 
ferent organs  in  the  body,  producing  in  them  more  or 
less  intense  inflammatory  processes,  which  are  mostly  of 
a  purulent  character.  It  may  thus  cause  inflammations 
of  the  serous  membranes  of  the  endocardium,  the  peri- 
cardium, the  meuinges,  and  even  of  the  brain  itself. 
Foremost  among  the  secondary  infections  which  it 
causes  are  meningitis,  serofibrinous  pleurisy,  and  em- 
pyema.  In  25  cases  of  purulent  meningitis  examined 
by  Netter  the  "  pneumococcus  "  was  found  in  16;  4  of 
these  cases  were  complicated  with  purulent  otitis,  6 
with  pneumonia,  and  3  with  ulcerative  endocarditis. 
In  45  cases  collected  by  Netter  from  the  literature  of 


MICROCOCCUS  LANCEOLATUS.  509 

the  subject  this  micrococcus  was  present  in  27.  Monti 
demonstrated  the  presence  of  the  same  micrococcus  in 
4  cases  of  cerebro-spinal  meningitis.  Weichselbaum, 
in  a  series  of  29  cases  of  ulcerative  endocarditis  exam- 
ined, found  <(  diplococcus  pneumoniae"  in  7.  It  has 
been  found  also  in  acute  abscesses — in  the  pus  of  paro- 
titis complicating  pneumonia  it  was  obtained  in  pure 
culture  by  Testi;  in  a  case  of  pneumonia,  in  which 
there  developed  a  purulent  pleuritis  and  parotitis,  and 
multiple  subcutaneous  abscesses,  it  was  found  in  great 
numbers  in  all  these  places  in  the  pus;  in  a  case  of 
tonsillitis  resulting  in  the  formation  of  an  abscess  it 
was  obtained  in  pure  culture  by  Gabbi.  This  micro- 
coccus  has  also  been  found  in  a  number  of  cases  of 
otitis  media — in  the  pus  obtained  by  paracentesis  of 
the  tympanic  membrane,  by  Netter  in  5  out  of  18  cases 
occurring  in  children.  Monti  and  Belfanti  report  cases 
of  arthritis  of  the  wrist-joint,  occurring  as  a  complica- 
tion of  pneumonia,  in  which  it  was  found.  Ortmann 
and  Samter,  in  a  case  of  purulent  inflammation  of  the 
shoulder-joint  following  pneumonia  and  pleurisy,  ob- 
tained the  u  pneumococcus  "  in  pure  culture.  It  has 
been  found  in  other  cases  of  inflammation  of  the  knee, 
ankle,  and  elbow-joints,  and  in  osteomyelitis  and  perios- 
titis. In  short,  there  is  scarcely  any  part  of  the  body 
in  which  this  organism  may  not  find  suitable  conditions 
for  existence  and  in  which  it  does  not  sometimes  occur. 
How  is  it  conveyed  from  its  original  seat  in  the  lungs 
to  distant  internal  organs?  Chiefly  by  means  of  the 
bloodvessels  and  lymphatics,  in  both  of  which  it  has 
been  found  in  great  numbers.  Proof  enough  of  its 
conveyance  through  the  lymphatics  is  afforded  by  the 
frequent  occurrence  of  inflammations  of  the  serous 


510  BACTERIOLOGY. 

membranes  complicating  pneumonia;  but  two  cases  in 
particular  have  been  reported  by  Thue  of  pleurisy  and 
pericarditis  following  pneumonia  in  which  the  lymph 
capillaries  have  been  found  to  be  chock-full  of  diplo- 
cocci,  as  if  injected.  Their  presence  in  the  blood  after 
death  has  been  amply  proved  by  numerous  investiga- 
tions. In  many  instances  they  have  been  recovered 
from  the  blood  during  life.  Lambert,  as  a  rule,  found 
them  in  all  fatal  cases  twenty-four  to  forty-eight  hours 
before  death.  This  examination  has  considerable  prog- 
nostic value,  as  nearly  all  cases  in  which  the  pneumo- 
coccus  is  found  end  fatally.  This  micrococcus  has 
been  shown  experimentally  to  be  capable  of  producing 
various  forms  of  septicaemia — local  phlegm  onous  in- 
flammations, peritonitis,  pleuritis,  and  meningitis.  A 
further  proof  of  the  transmission  of  this  organism  by 
means  of  the  blood  is  given  by  Foa  and  Bordoni- 
Uffreduzzi  in  their  investigations  into  intra-uterine 
infection  in  pneumonia  and  meningitis.  These  in- 
vestigators have  demonstrated  the  presence  of  the 
micrococcus  lanceolatus  in  foetal  and  placental  blood 
and  in  the  uterine  sinuses  in  maternal  pneumonia. 
There  being  no  question,  therefore,  as  to  the  pos- 
sibility of  the  conveyance  of  the  infective  agent  by 
means  of  the  blood  and  the  lymph  to  all  parts  of  the 
body,  we  need  not  wonder  at  the  multiplicity  of  the 
affections  complicating  pneumonia  which  are  caused  by 
this  micrococcus ;  and  not  only  the  secondary,  but  also 
the  primary  diseases,  as  of  the  brain  and  the  meninges, 
may  be  explained  in  the  same  way.  Knowing  that  the 
saliva  and  nasal  secretions  under  normal  conditions  so 
frequently  afford  a  resting-place  for  the  micrococci,  we 
have  only  to  assume  the  production  of  a  suitable  culture 


MICROCOCOUS  LANCEOLATUS.  511 

medium  for  these  parasites  in  the  body,  brought  about 
by  an  abnormal  condition  of  the  mucous  membranes 
from  exposure  to  cold,  or  a  reduction  of  the  vital  re- 
sisting power  of  the  tissue  cells  in  any  of  the  internal 
organs,  caused  by  disease,  traumatism,  excesses  of  vari- 
ous kinds,  etc. ,  to  readily  comprehend  how  an  individual 
may  become  infected  with  pneumonia,  either  primarily 
affecting  the  lungs  and  secondarily  other  organs  in  the 
body,  or  primarily  attacking  the  middle  ear,  the  pericar- 
dial  sac,  the  pleura,  the  serous  cavities  of  the  brain,  etc. 
From  statistics  collected  by  Netter  the  following  per- 
centages of  diseases  were  caused  by  the  ' '  diplococcus 
pneumonias7 ' : 

Pneumonia      'r-   .  .  • .     65.9  per  cent,  in  adults. 

Bronchopneurnonia  .  .     15.8        ' 

Meningitis.         .  .  .13.0        ' 

Empyema   .         .  .  .       8.5        '  " 

Otitis  media        .  .  .       2.4       '  " 

Endocarditis       .  .  .       1.2        '  " 

In  46  consecutive  pneumococcus  infections  in  chil- 
dren there  were  : 

Otitis  media         .  *  '  .        .        .  29  cases. 

Bronchopneumonia  .  .         .         .  12     " 

Meningitis  .         .  .  .         .         .  2     ll 

Pneumonia .         .  .  ...  1     " 

Pleurisy       .         .  .  .  1     " 

Pericarditis          .  .  .         ...  1     " 

Varieties  of  the  Micrococcus  Lanceolatus.  The  ubi- 
quity of  this  organism  and  the  irregularity  of  its 
behavior  under  varying  conditions  have  opened  a  wide 
field  of  discussion  among  bacteriologists.  As  com- 
monly found,  for  instance,  in  the  saliva  of  different 
healthy  individuals,  and  even  in  that  of  the  same  in- 
dividual at  different  times,  it  often  varies  in  virulence; 


512  BACTERIOLOGY. 

and  as  obtained  from  the  saliva  it  differs  again 
from  the  organism  when  occurring  in  pneumonia. 
When  grown  on  artificial  culture  media,  variations  in  its 
morphology  have  been  observed,  at  one  time  the  indi- 
vidual cells  being  oval  in  shape  and  united  in  pairs,  and 
then  surrounded  by  a  capsule;  at  other  times  spherical 
and  arranged  in  longer  or  shorter  chains,  like  strepto- 
cocci, and  then  being  without  a  capsule.  Variations 
in  virulence  have  also  been  noted,  both  in  the  animal 
body  in  different  stages  of  disease  and  when  grown  out- 
side the  body  on  artificial  culture  media.  These  great 
variations  in  biological  and  pathogenic  properties  have 
induced  some  investigators  to  believe  that  there  were 
several  distinct  species  of  this  organism. 

These  views,  however,  have  not  met  with  general 
acceptance.  In  an  exhaustive  investigation  into  the 
subject  by  Kruse  and  Pansini,  who  obtained  the  micro- 
cocci  from  the  most  varied  sources — from  the  saliva  in 
health  and  disease,  from  the  nasal  secretions,  from  pneu- 
monic sputum  at  different  periods  and  phases  of  the 
illness,  from  the  blood  of  different  kinds  of  animals 
killed  by  inoculation,  and,  finally,  from  many  primary 
and  secondary  affections  due  to  this  organism — after 
carefully  weighing  their  results  from  different  points 
of  view  and  comparing  the  morphological,  biological, 
and  pathological  characteristics  of  the  various  micro- 
cocci  found,  these  observers  have  come  to  the  conclu- 
sion that  "it  is  impossible  to  distinguish  different 
varieties"  of  pneumococci.  They  found  numerous 
quantitative  and  qualitative  variations  in  virulence, 
growth,  power  of  resistance,  etc.,  but  at  the  same  time 
such  an  inconstancy  in  these  variations  that  they  were 
unable  to  make  any  classification  into  separate  varieties. 


MICROCOCCUS  LANCEOLATUS.  513 

Judging  from  the  streptococcus,  the  use  of  a  specific 
bactericidal  serum  developed  from  a  single  pneumo- 
coccus  will  probably  show  that  some  of  the  organisms 
ranked  as  pneumococci  are  not  influenced  by  it. 

Immunity.  Early  in  the  history  of  this  organism 
experiments  were  begun  for  the  production  of  immunity 
in  animals  by  means  of  preventive  inoculations. 
Fraenkel  showed  that  subcutaneous  injections  of  rab- 
bits with  virulent  cultures  of  the  diplococcus  produced 
infection  in  only  a  small  percentage  of  these  animals, 
which  either  died  from  septicaemia  or  after  a  time  re- 
covered. In  the  latter  case  they  were  found  to  be  some- 
what immune  to  a  second  infection.  Later  experiments 
were  conducted  on  the  same  principle,  the  object  being 
to  repeatedly  slightly  infect  the  animal,  and  thus  to 
gradually  increase  its  power  of  resistance  to  infection. 
For  this  purpose  either  artificially  attenuated  cultures 
or  material  containing  naturally  attenuated  micrococci 
were  used  for  inoculation.  Cultures  artificially  attenu- 
ated by  heat  or  several  days'  growth  in  the  incubator, 
sputum  taken  from  a  pneumonic  patient  after  the  crisis, 
rusty  sputum  obtained  before  the  crisis  and  heated  to 
60°  C.,  old  pleuritic  exudation  containing  attenuated 
bacteria,  etc.,  have  thus  bejen  repeatedly  employed. 

Another  series  of  experiments  were  based  on  the 
assumption  that  the  immunizing  substances  are  con- 
tained in  the  natural  or  artificial  products  of  the  growth 
of  the  organism.  Thus  cultures  which  were  freed  from 
bacteria  by  filtration,  and  emulsions  of  pneumonic 
sputum,  portions  of  pneumonic  lung,  pleuritic  exuda- 
tions, etc.,  were  employed  by  different  experimenters. 
The  quantity  of  material  required  for  inoculation  being 
found  inconveniently  large,  attempts  were  then  made 

33 


514  BACTERIOLOGY. 

to  obtain  the  immunizing  substances  in  a  more  concen- 
trated form.  Foa  and  Scabia  and  F.  and  G.  Klein  - 
perer  prepared  glycerin  extracts,  after  the  manner  of 
Koch,  calling  their  extract  "  pneumoprotein."  At 
present,  however,  a  protective  serum  is  obtained  from 
horses  by  the  repeated  injections  of  fully  virulent 
pneumococci  in  exactly  the  same  manner  as  in  the  pro- 
duction of  antistreptococcus  serum. 

Therapeutic  Experiments.  Curative  experiments  in 
man  have  also  recently  been  made  with  the  blood-serum 
of  immunized  animals  and  of  persons  who  have  recov- 
ered from  an  attack  of  pneumonia.  The  most  success- 
ful of  these  were  conducted  by  F.  and  G.  Klemperer. 
These  authors  hold  that  in  man  during  the  pneumonic 
process  there  is  a  constant  absorption  into  the  circula- 
tion of  this  toxic  albuminous  substance  produced  by 
the  bacteria  in  the  lungs.  This  continues  until  event- 
ually the  same  antitoxic  substance  is  produced  in  the 
circulation  that  has  been  seen  to  occur  experimentally. 
It  is  then  that  the  crisis  occurs.  The  bacteria  are 
neither  destroyed  nor  is  their  power  to  produce  pneu- 
motoxin  lessened;  but  the  third  factor — the  antipneumo- 
toxin — now  exists  and  neutralizes  the  toxic  substances 
as  they  are  produced.  They  apparently  demonstrated 
that  the  serum  of  the  blood  of  patients  after  the  crisis 
of  pneumonia  contained  antitoxic  substance,  and  was 
capable,  in  a  fair  number  of  cases,  of  curing  the  dis- 
ease when  injected  into  infected  animals.  They  have 
made  preliminary  observations  upon  patients  with  a 
view  of  inducing  the  crisis  by  the  injection  of  the 
blood-serum  of  persons  convalescent  from  pneumonia, 
and  which,  consequently,  contain  the  antitoxic  body. 
In  six  pneumonic  patients  the  results  were  promising. 


MICROCOCCUS  LANCEOLATUS.  515 

In  all  there  was  a  decided  fall  of  temperature  in  from 
six  to  twelve  hours  after  subcutaneous  injections  of  from 
4  to  6  c.c.  of  the  serum.  The  pulse  and  respirations 
were  also  diminished  in  frequency.  In  two  cases  the 
temperature  fell  to  37°  C.  Twice  it  fell  and  remained 
at  normal.  In  other  cases  it  fell  only  temporarily. 

The  number  of  cases  reported  in  which  the  blood- 
serum  of  animals  artificially  immunized  against  pneu- 
monic infection  has  been  used  for  the  treatment  of  the 
disease,  although  considerable,  is  still  too  few  to  warrant 
the  expression  of  any  definite  opinion  as  to  the  final 
value  of  this  as  a  therapeutic  agent.  In  the  cases  we 
have  observed  there  has  been  in  some  a  slight  imme- 
diate lowering  of  the  temperature  ;  in  others  no  ap- 
parent change.  As  a  rule,  the  cases  did  rather  better 
than  was  expected,  but  certainly  no  striking  curative 
effects  were  apparent.  The  cases  did  not  develop 
pnetimococcus  blood  infection,  and  it  seems  probable 
that  the  serum  may  be  able  to  prevent  a  general  infec- 
tion from  taking  place  from  the  diseased  lung,  even 
though  it  may  fail  to  influence  the  local  process.  It 
has  also  been  shown  that  these  injections  of  antipneu- 
motoxic  serum  are  practically  harmless. 


CHAPTER  XXIX. 

DIPLOCOCCUS   INTRACELLULARIS   MENINGITIDIS. 

IN  the  description  of  the  micrococcus  lanceolatus 
reference  was  made  to  this  organism  as  the  most  fre- 
quent cause  of  meningitis,  especially  when  it  compli- 
cated pneumonia.  In  1887,  Weichselbaum  discovered 
another  micrococcus  in  the  exudate  of  cerebro-spinal 
meningitis  in  six  cases,  two  of  which  were  not  com- 
plicated by  pneumonia.  He  obtained  it  in  pure  cultures, 
studied  its  characteristics,  and  showed  that  this  organism 
was  clearly  distinguishable  from  the  micrococcus  lanceo- 
latus, and  especially  by  its  usual  presence  in  the  interior 
of  pus-cells,  on  which  account  he  called  it  diplococcus 
intracellularis  meningitidis.  The  frequency  of  the  occur- 
rence of  this  diplococcus  in  meningitis  and  its  restric- 
tion to  this  disease  affords  sufficient  evidence  for  the 
assumption  that  it  was  concerned  in  its  production. 
In  1895,  Jaeger  and  Scheurer  found  it  in  the  nasal 
secretions  of  eighteen  living  persons  suffering  from  this 
disease  during  an  epidemic. 

Morphology.  This  organism  occurs  as  biscuit-shaped 
micrococci,  usually  united  in  pairs,  but  also  in  groups 
of  four  and  in  small  masses;  sometimes  solitary  and 
smaller  degenerated  forms  are  found.  In  the  exuda- 
tion, like  the  gonococcus,  to  which  it  bears  a  close  re- 
semblance in  form  and  arrangement,  it  is  distinguished 
by  its  presence  within  the  polynuclear  leucocytes.  It 


INTRA  CELL  ULARIS  MENINGITIDIS.          517 

never  appears  within   the  nucleus  and  rarely  within 
other  cells  (Fig.  67). 

It  stains  with  all  the  ordinary  aniline  colors,  but  best 
with  Loffler's  methylene-blue.  According  to  Weich- 
selbaum,  it  is  decolorized  by  Gram's  solution;  Jaeger 
states  that  this  is  not  constantly  the  case. 

FIG.  67. 


Diplococcus  intracellularis  meningitidis.    X  1100  diameters. 

Biological  Characters.  It  does  not  grow  at  room-tem- 
perature, but  only  in  the  incubator,  and  its  develop- 
ment is  usually  scanty  on  the  surface  of  agar,  but  some- 
times a  few  colonies  grow  quite  vigorously.  It  does 
not  grow  at  all  or  scarcely  any  in  bouillon,  and  very 
scantily  in  bouillon  plus  one-third  blood-serum.  It 
grows  comparatively  well  on  Loffler's  blood-serum 
medium  as  used  for  diphtheria  cultures. 

When  grown  on  nutrient  agar  or  glycerin-agar,  at 
the  end  of  forty-eight  hours  in  the  incubator,  there 
develops  a  tolerably  good  growth,  appearing  as  a  flat 
layer  of  colonies,  about  one-eighth  of  an  inch  in  diam- 


518  BACTERIOLOGY. 

eter,  grayish- white  in  color,  viscid  and  non-confluent 
unless  very  close  together.  On  Loffler's  blood-serum 
the  growth  forms  round,  whitish,  shining  viscid-look- 
ing colonies,  with  smooth  and  sharply-defined  outlines, 
and  may  attain  diameters  of  one-eighth  to  one-sixteenth 
of  an  inch  in  twenty-four  hours.  The  colonies  tend  to 
become  confluent  and  do  not  liquefy  the  serum.  In 
acute  cases,  where  the  organisms  are  apt  to  be  more 
abundant,  a  great  many  minute  colonies  may  develop 
instead  of  a  few  larger  ones.  On  agar  plates  the 
deep-lying  colonies  are  almost  invisible  to  the  naked 
eye;  somewhat  magnified  they  appear  as  finely  granular 
colonies,  with  a  dentated  border.  On  the  surface  they 
are  larger,  appearing  as  pale  disks,  almost  transparent 
at  the  edges,  but  more  compact  toward  the  centres, 
which  are  yellowish -gray  in  color.  Cultivated  in  arti- 
ficial media  it  soon  loses  its  vitality — within  six  days 
— and  requires,  therefore,  to  be  transplanted  to  fresh 
material  at  short  intervals — at  least  every  two  days. 

Pathogenesis.  This  organism  does  not  show  much 
pathogenic  power  for  animals.  It  is  most  pathogenic 
for  mice  and  guinea-pigs,  less  so  for  rabbits  and  dogs. 
Subcutaneous  injections  in  animals  give  negative  re- 
sults; intrapleural  or  intraperitoneal  inoculations  in 
mice  and  guinea-pigs,  when  given  in  large  doses,  are 
usually  successful.  Intravenous  injections  in  rabbits 
have  caused  the  death  of  the  animal,  but  no  diplococci 
or  pathological  changes  have  been  found  as  a  result  of 
the  injections. 

When  mice  are  inoculated  into  the  pleural  or  peri- 
toneal cavities  they  usually  fall  sick  and  die  within 
thirty-six  to  forty-eight  hours,  showing  slight  fibrino- 
purulent  exudation.  In  the  blood  and  enlarged  spleen 


INTRA  CELL  ULARIS  MENINGITIDIS.          519 

.  diplococci  are  found  in  small  numbers  and  mostly  free; 
in  the  pleuritic  exudation  they  are  present  in  consider- 
able quantities,  less  so  in  the  peritoneal  fluid,  but  then 
occurring  in  the  interior  of  pus-cells. 

Certain  experiments  made  by  Weichselbaum  on  dogs, 
though  not  entirely  successful,  are  interesting  as  showing 
the  similarity  of  the  disease  produced  in  them  artificially 
with  meningitis  as  occurring  in  man.  The  three  dogs, 
trephined  and  inoculated  subdurally  with  0.5  to  2  c.c.  of 
afresh  culture,  all  died:  No.  1  within  twelve  hours,  No. 
2  in  three  days,  and  No.  3  in  twelve  days.  In  Nos.  1 
and  2  there  were  found  hypersemia  of  the  meninges,  with 
inflammatory  softening  of  the  brain  at  the  point  of  inocu- 
lation, which  on  nearer  inspection  proved  to  be  a  true 
encephalitic  process.  In  dog  No.  2,  in  which  the  dis- 
ease was  of  longer  duration,  these  changes  were  the 
most  pronounced.  Numerous  diplococci  were  observed 
in  the  sections  removed,  for  the  most  part  free,  but 
some  few  within  the  pus-cells.  In  dog  No.  3,  in 
which  the  disease  lasted  twelve  days,  between  the  dura 
mater  and  the  brain,  at  the  point  of  inoculation,  was 
found  a  thick,  reddish,  purulent  liquid;  in  the  brain 
itself  an  abscess  had  formed,  about  the  size  of  a  hazel- 
nut,  filled  with  tough,  yellow  pus,  while  the  abscess 
walls  consisted  of  softened  brain -substance  infiltrated 
with  numerous  hemorrhagic  deposits,  and  simultane- 
ously the  ventricles  on  that  side  contained  a  cloudy, 
reddish  fluid,  with  flocks  of  pus;  but  no  diplococci 
could  be  demonstrated  in  the  blood  or  exudations. 
Weichselbaum  suggests  that  under  natural  conditions 
the  diplococci  gain  access  to  the  brain  and  meninges  by 
way  of  the  nose,  ear,  and  upper  air-passages.  Cerebro- 
spinal  meningitis,  as  is  well  known,  is  often  accorn- 


520  BACTERIOLOGY. 

panied  by  rhinitis  and  purulent  inflammation  of  the 
mucous  membranes  of  the  nose.  In  one  of  his  six  cases 
Weichselbaum  succeeded  in  obtaining  in  pure  culture 
diplococci  from  the  nasal  secretion.  Scheurer,  in  his 
eighteen  cases,  found  the  diplococci  in  the  nasal  secre- 
tions of  all  of  them  during  life.  In  fifty  healthy  in- 
dividuals examined  they  were  found  in  the  nasal  secre- 
tions of  only  two  of  them,  one  being  a  man  suffering 
at  the  time  from  a  severe  cold.  This  man,  it  is  inter- 
esting to  note,  had  been  engaged  in  disinfecting  a  room 
which  had  previously  been  occupied  by  a  patient  with 
cerebro-spinal  meningitis. 

Bacteriological  Diagnosis.  By  means  of  lumbar  punc- 
ture fluid  can  be  readily  obtained  without  danger  from 
the  spinal  canal.  The  microscopical  examination  will 
frequently  reveal  numerous  cells  crowded  with  diplo- 
cocci. When  considerable  quantities  are  inoculated 
upon  Loffler's  blood-serum  mixture  or  upon  glycerin 
agar,  as  a  rule,  a  greater  or  less  number  of  colonies 
having  the  characteristics  already  described  will  de- 
velop. The  value,  clinically,  of  the  examination  is 
that  about  40  per  cent,  of  the  cases  due  to  this  coccus 
recover,  while  almost  all  of  those  due  to  the  pneumo- 
coccus  and  streptococcus  die.  In  fifty-five  cases  exam- 
ined by  Councilman,  Mallory,  and  Wright,  diplococci 
were  found  in  the  fluid  removed  by  lumbar  puncture  in 
thirty-eight,  either  by  microscopical  examination  or 
cultures. 

The  longest  time  after  the  onset  of  the  disease  in  which 
positive  results  were  obtained  by  culture  was  twenty- 
nine  days.  In  a  number  of  cases  examined  by  us  for 
Northrup  a  rather  smaller  percentage  of  the  cases  were 
found  to  be  due  to  this  diplococcus.  In  many  cases 


INTEA  CELL  ULAEIS  MENINGITIDIS.          521 

there  are  very  few  diplococci  present  in  the  spinal  fluid, 
so  that  a  failure  to  find  them  in  a  microscopical  examina- 
tion should  not  be  taken  to  prove  that  the  disease  was 
not  due  to  this  organism.  For  cultures  a  considerable 
amount  of  fluid  must  be  used,  for  we  have  found,  as 
described  by  Councilman  and  others,  that  there  may 
be  very  few  living  diplococci  even  in  1  c.c.  of  fluid. 

To  obtain  the  fluid  the  patient  should  lie  on  the  right 
side  with  the  knees  drawn  up  and  the  left  shoulder  de- 
pressed. The  skin  of  the  patient's  back,  the  hands  of 
the  operator,  and  the  large  antitoxin  syringe  should  be 
sterile.  The  needle  should  be  4  cm.  in  length,  with  a 
diameter  of  1  mm.  for  children,  and  longer  for  adults. 

The  puncture  is  generally  made  between  the  third 
and  fourth  lumbar  vertebrae.  The  thumb  of  the  left 
hand  is  pressed  between  the  spinous  processes,  and  the 
point  of  the  needle  is  entered  about  1  cm.  to  the  right 
of  the  median  line  and  on  a  level  with  the  thumb-nail, 
and  directed  slightly  upward  and  inward  toward  the 
median  line.  At  a  depth  of  3  or  4  cm.  in  children  and 
7  or  8  cm.  in  adults  the  needle  enters  the  subarachnoid 
space,  and  the  fluid  flows  out  in  drops  or  in  a  stream. 
If  the  needle  meets  a  bony  obstruction  withdraw  and 
thrust  again  rather  than  make  lateral  movements. 
Any  blood  obscures  the  microscopical  examination. 
The  fluid  is  allowed  to  drop  into  absolutely  sterile  test- 
tubes  or  vials  with  sterile  stoppers.  From  5  to  15  c.c. 
should  be  withdrawn.  No  ill  effects  have  been  ob- 
served from  the  operations. 


CHAPTER  XXX. 

MICROCOCCUS  GONORRHOEA  (OONOCOCCUS  NEISSER). 

THIS  micrococcus  was  first  observed  by  Neisser,  in 
1879,  in  gonorrhoeal  discharges,  and  described  by  him 
under  the  name  of  "  gonococcus";  but  though  several 
attempted  to  discover  a  medium  upon  which  it  might 
be  cultivated,  it  was  reserved  for  Bumm,  in  1885,  to 
obtain  it  in  pure  culture  and  isolate  it  and  then  prove 
its  infective  virulence  by  inoculation  into  man.  Since 
that  time  the  gonococcus  has  been  cultivated  on  various 
media,  which,  though  modifications  of  Bumm's,  are  an 
improvement  on  his  original  method,  and  as  the  result 
of  various  inoculation  experiments  there  now  remains 
no  doubt  that  this  organism  is  the  specific  cause  of 
gonorrhoea  in  man. 

Morphological  Characters.  Micrococci,  occurring 
mostly  in  the  form  of  diplococci — that  is,  in  pairs  or 
in  groups  of  four.  The  bodies  of  the  diplococci  are 
elongated,  and,  as  shown  in  stained  preparations,  have 
an  unstained  division  or  interspace  between  two  flat- 
tened surfaces  facing  one  another,  which  gives  them 
their  characteristic  "  coffee-bean "  or  "biscuit"  shape 
(Fig.  68).  The  diameter  of  an  associated  pair  of  cells 
varies  from  0.8//  to  1.6//  in  the  long  diameter — 
average  about  1.25// — by  0.6/*  to  0.8/u.  in  the  cross 
diameter.  In  gonorrhoea  gonococci  are  found  mostly 
in  small,  irregular  groups  in  or  upon  the  pus-cells,  and 


MIGROGOGGUS  GONORRHCEJE.  523 

generally  extranuclear.  When  found  in  other  portions 
of  the  field  this  is  mostly  due  to  the  mechanical  effect 
of  smearing  the  pus  on  cover-glass  slides,  and  should 
not  be  considered  as  characteristic.  That  the  gonococci 
really  lie  within  the  protoplasm  of  the  cells  is  proved 
by  the  fact  that  in  carefully  made  preparations  they 
are  usually  not  found  outside  of  the  pus-cells.  They 
appear  usually  as  diplococci,  in  groups  of  two  or  four, 

FIG.  68. 


Smear  from  pure  culture  of  gonococcus  on  agar.    (HEIMAN.) 

but  at  times  they  occur  as  round,  single,  and  undivided 
cells.  Others,  again,  are  irregular  in  shape  or  granular 
in  appearance,  involution  forms,  particularly  in  older 
cultures  and  in  chronic  urethritis  of  long  standing. 
The  pus-cells  containing  gonococci  are  most  numerous 
in  the  later  or  purulent  stage  of  the  disease,  not  so  fre- 
quent in  the  beginning  of  infection,  or  as  long  as  the 
discharge  is  of  a  serous  character  (Fig.  69). 

The  gonococcus  stains  readily  with  the  basic  aniline 
colors,  especially  with  methyl-violet,  gentian-violet,  and 


524  BACTERIOLOGY. 

fuchsin;  not  so  readily  with  methylene-blue,  which  is, 
however,  one  of  the  best  staining  agents  for  demon- 
strating its  presence  in  pus.  Beautiful  double-stained 
preparations  may  be  made  from  gonorrhoeal  pus  by 
treating  cover-glass  smears  with  methyl-violet  and 
eosin.  Gonococci  are  decolorized  by  Gram's  solution; 
but  this  cannot  be  depended  upon  alone  to  absolutely 
distinguish  the  gonococcus  from  all  other  diplococci 

FIG.  69. 


Gonococcus  in  pus-cells.    X  1100  diameters. 

found  in  the  urethra  and  vulvo-vaginal  tract,  for  espe- 
cially in  the  female  other  diplococci  are  occasionally 
found  which  are  also  not  stained  by  Gram's  method. 
It  serves,  however,  to  distinguish  this  micrococcus  from 
the  common  pyogenic  cocci,  which  retain  their  color 
when  treated  in  the  same  way,  and  in  the  male  urethra 
it  is  practically  certain,  as  no  organism  has  been  found 
in  that  location  which  in  morphology  and  staining  is 
identical  with  the  gonococcus.  It  is  certainly  the  most 
distinctive  characteristic  of  the  staining  properties  of 


MICROCOCCUS  GONORRHCEjE.  525 

the  gonococcus,  and  it  is  a  test  that  should  never  be 
neglected  in  differentiating  this  organism  from  others 
which  are  morphologically  similar. 

Biological  Characters.  The  elaborate  experiments  of 
Bumm  and  others  have  shown  that  at  the  ordinary  room- 
temperature  no  growth  of  the  specific  micrococcus  occurs 
on  the  culture  media.  Apparently  positive  results  which 
have  been  reported  are  found  to  be  due  to  other  diplo- 
cocci  morphologically  almost  identical  with  the  gono- 
coccus. 

Since  Bumm's  experiments  a  number  of  culture 
methods  have  been  proposed  for  the  gonococcus  which 
are  an  improvement  on  Bumm's,  partly  because  the 
growth  produced  is  more  constant  and  luxuriant  and 
partly  because  the  media  employed  are  more  readily 
prepared.  Wertheim  (1892)  succeeded  in  developing 
luxuriant  and  virulent  cultures  to  many  generations  on 
a  mixture  of  placenta  blood-serum  and  2  per  cent,  pep- 
tone-agar.  His  method  is  briefly  as  follows  :  Several 
loops  of  gonorrhoeal  pus  are  diffused  through  liquid 
blood-serum  warmed  to  40°  C.  contained  in  a  test- 
tube.  Two  dilutions  are  made  from  this,  and  an 
equal  quantity  of  melted  2  per  cent,  agar  cooled  to 
40°  C.  is  added  to  the  three  tubes,  and  the  contents, 
after  thorough  mixing,  poured  into  Petri  dishes.  The 
Petri  dishes  are  placed  in  an  incubating  oven  at  a 
temperature  of  36°  to  37°  C.  At  the  end  of  twenty- 
four  hours  there  will  have  developed  on  at  least  one  of 
the  plates  distinct  colonies;  these  are  translucent,  finely 
granular,  with  scalloped  margin.  By  transferring  such 
a  colony  to  slant-cultures  of  serum-agar,  pure  cultures 
of  the  gonococcus  are  obtained;  these  are  somewhat 
shining  in  appearance  and  of  a  grayish-white  color. 


526  BACTERIOLOGY. 

Wertheim  demonstrated  that  the  addition  of  peptone 
to  the  culture  medium  was  an  important  factor  in  the 
cultivation  of  gonococci.  Kiefer  (1895)  proposed  a  cul- 
ture medium  consisting  of  one  part  of  hydrothorax  or 
ascitic  fluid  and  one  part  of  a  fluid  containing  3.5  per 
cent,  agar,  5  per  cent,  peptone,  2  per  cent,  glycerin,  and 
0.5  per  cent.  salt. 

Simultaneously  with  Kiefer,  Heiman  recommended 
a  medium  made  from  hydrocele  fluid,  or  from  "  chest- 
serum"  obtained  from  a  patient  suffering  from  hy- 
drocele or  hydrothorax  or  acute  pleurisy.  Having 
experimented  with  all  the  various  culture  media  here- 
tofore prepared  for  the  cultivation  of  the  gonococctis, 
Heiman  believes  this  medium  to  be  superior  to  placenta - 
serum,  sheep  blood-serum,  or  ascitic  fluid,  because  of 
the  large  amount  of  serum  albumin  which  it  contains. 
The  medium  consists  of  a  2  per  cent,  agar,  plus  2  per 
cent,  peptone,  plus  0.5  per  cent,  salt  and  2  per  cent, 
glucose.  Of  this  mixture  two  parts  are  added  to  one 
part  of  chest -serum,  which  is,  if  necessary,  fractionally 
sterilized  between  65°  and  70°  C.  for  one  hour  for  seven 
consecutive  days.  The  chest-serum-agar  should  have  a 
neutral  reaction.  The  growth  on  this  medium  is  thus 
described  :  In  plate  cultures  streaked  on  the  surface, 
growth  abundant,  colonies  circular  in  shape,  edges 
somewhat  irregular,  shading  off  into  yellowish- white; 
texture  finely  granular  in  periphery,  presenting  punc- 
tated spots  of  higher  refraction  in  and  around  the 
centre  of  yellowish  color  (Fig.  70). 

The  gonococcus  has  but  little  resistant  power  against 
outside  influences.  It  is  killed  by  weak  disinfecting 
solutions  and  by  desiccation  in  thin  layers.  In  com- 
paratively thick  layers,  however,  as  when  gonorrhoeal 


MICROCOCCUS  GONORRHCE^E. 


527 


pus  is  smeared  on  linen,  it  has  lived  for  forty-nine 
days,  and  dried  on  glass  for  twenty-nine  days  (Heiman). 
No  development  takes  place  below  25°  C.  or  above 
39°  C.;  it  is  killed  at  a  temperature  over  42°  C. 

Pathogenesis.  Non-transmissible  to  dogs,  monkeys, 
horses,  and  rabbits,  whether  inoculations  be  made  into 
the  urethral,  vaginal,  or  congenital  mucous  membranes. 
According  to  Wertheim,  purulent  peritonitis,  not  caus- 

FIG.  70. 


Colonies  of  gonococci  on  pleuritic  fluid  agar.    (HEIMAN.) 

ing  death,  is  produced  in  certain  animals  by  the  intro- 
duction of  pieces  of  serum-agar  containing  colonies  of 
the  gonococcus.  This  effect  was  produced  constantly  in 
mice,  occasionally  in  guinea-pigs,  and  rarely,  if  ever, 
in  dogs,  rats,  and  rabbits. 

Though  animal  inoculations  are  thus  followed  by 
negative  results,  the  etiological  relation  of  the  gono- 
coccus to  human  gonorrhoea  has  been  demonstrated 
beyond  question  by  the  infection  of  healthy  men  with 
the  disease  by  inoculation.  Thus,  Bumm  has  produced 


528  BACTERIOLOGY. 

gonorrhoea  in  normal  urethral  mucous  membranes  by 
inoculation  of  a  pure  culture  on  blood-serum  in  the 
second  generation;  Wertheim,  in  the  thirtieth;  Kiefer, 
in  the  sixth,  and  Heiman  in  the  fifth  generation.  At 
the  same  time  the  distinctive  morphological,  staining  and 
biological  characters  of  the  organism  were  carefully  noted 
and  confirmed  to  be  those  of  the  gonococcus  by  these  ob- 
servers; the  typical  incubation  and  symptoms  of  the  dis- 
ease resulted  in  all  cases  in  the  subjects  experimented  on. 
According  to  the  observations  of  the  most  reliable 
investigators  and  those  most  familiar  with  the  various 
forms  of  micrococci  which  are  likely  to  be  mistaken 
for  the  gonococcus,  affections  due  to  this  organism  are 
usually  restricted  to  the  mucous  membranes  of  the 
urethra,  conjunctiva,  bladder,  cervix  uteri,  and  rectum. 
It  rarely,  if  ever,  produces  a  vaginitis  in  adults;  but 
occasionally  a  vulvo- vaginitis  in  young  children.  For- 
merly the  presence  of  gonococci  could  only  be  deter- 
mined microscopically;  but  since  the  introduction  of  the 
serum-agar  the  culture  method  has  rendered  the  diag- 
nosis of  gonorrhoea  much  more  reliable.  This  method 
of  investigation,  moreover,  has  given  valuable  infor- 
mation with  regard  to  the  nature  of  many  infections 
complicating  or  resulting  from  gonorrhoea,  particularly 
in  affections  of  the  uterus  and  joints,  about  which  there 
was  heretofore  considerable  doubt,  though  the  micro- 
cocci  often  found  in  these  organs  were  morphologically 
identical  with  the  gonococcus.  It  has  now  been  shown 
by  the  culture  method  that  gonococci  may  occur  in  the 
joints  in  gonorrhoeal  arthritis,  in  the  Fallopian  tubes  in 
salpingitis,  and  in  ovarian  abscesses;  and  Wertheim 
asserts  that  he  has  found  them  in  the  infiltrated  con- 
nective tissue  in  parametritis. 


MICROCOCCUS  GONOREHCEjE.  529 

Worthy  of  special  notice  in  this  connection  are  the 
cases  of  endocarditis  accompanying  gonorrhoea,  and  some- 
times terminating  fatally,  when  they  are  known  as  en- 
docarditis gonorrhoeica  maligna.  The  question  naturally 
arises  in  these  cases  whether  it  is  the  gonococcus  or 
some  other  coccus  or  diplococcus  which  has  infected 
the  endocardium.  Here,  again,  it  is  only  by  means  of 
the  culture  method  that  this  question  can  be  settled 
definitely.  Fliigge  draws  attention  to  this  matter,  and 
states  that  but  few  cases  have  been  recorded  in  which 
the  information  given  as  to  the  cause  of  the  disease  can 
be  unhesitatingly  accepted.  Weichselbaum  mentions 
a  case  of  endocarditis  accompanying  gonorrhoea  which 
was  shown  by  the  culture  method  to  be  due  to  strepto- 
coccus infection,  proving  that  so-called  gonorrhoeal 
endocarditis  may  be  a  secondary  infection.  Other  cases 
are  recorded  by  Leyden,  Hiss,  Councilman,  and  Wilms 
which  are  said  to  have  been  most  probably  of  gonor- 
rhoeal  origin;  but  in  these  only  microscopical  examina- 
tions were  made  and  no  culture  experiments,  or  only 
cultures  on  gelatin  plates,  etc.,  which  were  inadequate. 
Welch  also  reports  a  case  of  endocarditis  with  general 
septicaemia  following  gonorrhoea,  in  which  he  demon- 
strated the  gonococcus  in  the  blood  of  a  living  person 
in  cover-glass  and  culture  medium.  No  other  patho- 
genic bacteria  were  found. 

Immunizing  Serum.  As  animals  are  not  infected  by 
the  gonococcus  they  are  not  very  suitable  for  injections 
with  the  cultures  for  the  purpose  of  producing  an  anti- 
toxic or  bactericidal  serum.  Their  insusceptibility  pre- 
sents also  an  almost  insurmountable  obstacle  to  the 
testing  of  the  blood  of  animals  under  treatment,  so  that 
although  it  may  be  possible  to  bring  about  an  artificial 

34 


530  BACTERIOLOGY. 

immunity  against  gonorrhoeal  infection,  information  on 
this  subject  is  at  present  wanting.  Immunity  in  man 
seems  to  be  similar  to  that  produced  after  infection  with 
the  other  pyogenic  cocci — that  is,  only  slight  in  amount 
and  for  a  short  period.  It  is  known  that  a  urethra  or 
cervix  may  contain  gonococci  which  lie  dormant  and 
may  be  innocuous  in  that  person  for  years,  but  which 
may  at  any  time  excite  an  acute  gonorrhoea  in  the  one 
carrying  the  infection  or  in  another  person. 

The  Bacteriological  Diagnosis  of  Gonorrhoea.  In  view 
of  the  fact  that  several  non-specific  forms  of  urethritis 
exist,  and  also  that  micrococci  morphologically  similar 
to  the  gonococcus  Neisser  are  often  found  in  the  normal 
urethral  and  vulvo-vaginal  tract,  it  becomes  a  matter 
of  great  importance  to  be  able  to  detect  gonococci  when 
present  and  to  differentiate  these  from  the  non-specific 
organisms.  The  gonococci  also  which  occur  in  old  cul- 
tures and  in  chronic  urethritis  of  long  standing  often 
take  on  a  very  diversified  appearance — sometimes 
nothing  but  an  irregular,  granular  mass  being  seen, 
which  renders  their  detection  difficult.  From  a  medico- 
legal  and  social  stand-point,  therefore,  the  differential 
diagnosis  of  the  gonococcus  has  in  certain  cases  a  very 
practical  significance. 

There  are  two  methods  of  differential  diagnosis  now 
available — the  microscopical  and  the  cultural.  Animal 
inoculations  are  of  little  value,  as  they  are  not  sus- 
ceptible, and,  of  course,  human  inoculations  are,  except 
in  extremely  important  cases,  generally  impossible.  In 
the  microscopical  diagnosis  it  should  be  borne  in  mind 
that  the  specific  gonococci  in  carefully  made  prepara- 
tions are  found  always  largely  within  the  pus-cells. 
Diplococci  morphologically  similar  to  gonococci  occur- 


MICROCOCCUS  GONOERHCE^.  531 

ring  in  other  portions  of  the  field  and  outside  of  the 
pus-cells  should  not  be  considered  specific  by  this  test 
only.  It  should  also  be  remembered  that  thegonococci 
are  decolorized  by  Gram's  method,  while  other  similar 
micrococci  which  occur  in  the  urethra  are,  as  a  rule, 
at  least,  not  so  decolorized.  Organisms  having  these 
characteristics  can  for  all  practical  purposes  be  con- 
sidered as  certainly  gonococci  if  obtained  from  the 
urethra.  From  the  vulvo-vaginal  tract  the  certainty  is 
not  so  great;  here  cultures  should  also  be  made.  Burnm, 
Heiman  and  others  have  shown  that  other  diplococci 
are  occasionally  found  in  gouorrhoeal  pus  from  the  vulvo- 
vaginal  tract,  and  very  rarely,  indeed,  from  the  urethra, 
which  do  not  stain  by  this  method.  Cover-glass  prepa- 
rations from  subacute  or  chronic  cases  should  be  ex- 
amined, if  possible,  with  a  microscope  provided  with  a 
mechanical  stage,  and  should  always  be  stained  by 
Gram's  method  and  the  examination  repeated  on  three 
consecutive  days.  Should  these  specimens  prove  nega- 
tive, to  exclude  any  possible  doubt  in  the  matter, 
cultures  should  then  be  made  on  chest-serum-agar, 
poured  in  dishes,  as  proposed  by  Heiman,  also,  if  with 
negative  results,  on  three  consecutive  days.  His 
method  of  procuring  the  urine  in  chronic  urethritis 
is  to  allow  the  patient  to  void  his  urine  either  imme- 
diately into  two  sterilized  centrifugal  tubes  or  first 
into  two  sterile  bottles.  The  first  tube  will  contain 
threads  of  the  anterior  urethra;  the  second  tube  will 
be  likely  to  contain  secretion  from  the  posterior  urethra 
and  from  the  prostate  gland  if,  while  urinating,  the 
patient's  prostate  be  pressed  upon  with  the  finger. 
Tubes  containing  such  urine  are  placed  in  the  centri- 
fuge and  whirled  for  three  minutes  at  twelve  hundred 


532  BACTERIOLOGY. 

or  more  revolutions  per  mintue;  the  threads  are  thrown 
down.  The  "  centrifuged"  sediment  will  be  found 
to  contain  most  of  the  bacteria  present,  epithelial  cells, 
and  at  times  spermatozoa.  Normal  urine  on  being 
"  centrifuged"  at  this  velocity  will  be  found  at  times 
slightly  turbid  at  the  bottom  of  the  tube.  This  tur- 
bidity will  be  found,  on  microscopical  examination,  to 
consist  of  epithelial  cells,  a  few  leucocytes,  and  some 
bacteria. 

Heiman  looks  upon  the  decolorization  by  Gram's 
method  as  the  only  reliable  criterion,  so  far  as  known, 
for  the  gonococcus  in  discharges  from  the  mucous  mem- 
branes, and  it  is  of  material  help,  also,  in  determining 
whether  a  culture  is  or  is  not  that  of  the  gonococcus. 
The  careful  examination  of  gonorrhoeal  threads  with 
cover-glass  by  Gram's  method  is  a  very  tedious  affair, 
as  in  every  instance  no  less  than  three  cover-glass  prep- 
arations should  be  looked  over  before  the  absence  of 
the  gonococcus  is  proved.  It  would  require  many 
hours  upon  each  and  every  specimen,  especially  if  the 
gonococci  are  present  in  very  small  number,  before  a 
reliable  and  conscientious  opinion  could  be  rendered. 
If,  after  all,  a  negative  opinion  is  ventured,  we  still 
are  under  the  necessity  of  proving  that  because  the 
threads  which  we  fished  out  for  the  cover-glass  exami- 
nation were  free  from  gonococci  the  remaining  ones 
were  also.  For  this  reason  the  culture  medium  is  more 
sensitive  for  bacteria  than  is  the  cover-glass,  for  we  are 
able  to  plant  each  and  every  thread  of  the  sediment  in 
the  centrifugal  tube.  Fiirbringer,  in  his  work,  men- 
tions the  fact  that  in  certain  cases  the  absence  of  the 
gonococcus  in  many  examinations  of  cover-glass  prep- 
arations is  not  a  positive  proof  that  the  gonococcus  is 


MICEOCOCCUS  GONORRHCEJE.  533 

not  present.  Heiman  was  able  to  confirm  the  correct- 
ness of  the  above  allusion,  for  on  one  occasion,  in 
examining  threads,  when  he  could  not  demonstrate  the 
gonococcus  in  cover-glass  preparations,  he  succeeded  in 
growing  it  on  chest-serum-agar  plates,  while  in  all 
instances  in  which  he  found  the  gonococcus  in  threads 
in  cover-glass  preparations  he  invariably  succeeded  in 
growing  it  on  chest-serum-agar  plates.  The  culture 
methods,  of  course,  presuppose  that  one  has  the  facili- 
ties and  knowledge  to  carry  them  out  successfully,  other- 
wise the  microscopical  methods  are  to  be  used  alone. 

In  acute  cases  the  specimen  for  examination  may  be 
collected,  when  the  patient  is  before  one,  by  passing  a 
sterilized  platinum  wire  loop  as  far  up  into  the  urethra 
as  possible  and  withdrawing  some  of  the  secretion. 
This  is  a  far  less  satisfactory  method  than  that  sug- 
gested by  Heiman,  by  "  centrifuging,"  except  when 
the  pus  is  abundant. 

The  Frequency  with  which  Gonococci  are  Found  in 
Smears  or  Cultures  in  Cases  of  Chronic  Urethritis. 
Heiman  found  in  61  cases  14  by  cultures  and  13  by 
smears.  The  following  results  were  obtained  by  other 
observers  by  cover-glass  preparations  :  Goll,  accord- 
ing to  his  elaborate  article,  examined  1046  cases  of 
chronic  urethritis  varying  in  duration  between  four 
weeks  to  six  years  or  more,  finding  gonococci  in  178 
cases,  the  remainder  giving  negative  results.  Neisser, 
out  of  143  cases  varying  in  duration  between  two 
months  and  eight  years,  found  gonococci  in  80  cases. 
Weinrich,  out  of  25  similar  cases,  obtained  2  positive 
results.  E.  Noeggerath,  in  1887,  deplored  the  fact 
that  on  account  of  the  lack  of  culture  media  for 
the  gonococcus  we  cannot  always  demonstrate  them. 


534  BACTERIOLOGY. 

Brose,  in  1893,  stated  that  the  culture  medium  is  the 
only  reliable  agent  for  the  detection  of  the  gonococcus. 
This  latter  statement  is  certainly  applicable  to  chronic 
urethritis  of  the  male.  Neisser,  in  1893,  stated  that 
in  chronic  urethritis  with  slight  discharge  the  examina- 
tion with  a  culture  medium  for  gonococci  will  replace 
the  cover-glass. 


CHAPTER  XXXI. 

BACILLUS  PYOCYANEUS  (BACILLUS  OF  GREEN  AND 
OF  BLUE  PUS)  —  BACILLUS  PROTEUS  VULGARIS 
— BACILLUS  OF  MALIGNANT  (EDEMA — BACILLUS 
AEROGENES  CAPSULATUS. 

BACILLUS  PYOCYANEUS. 

THE  blue  and  green  coloration  which  is  occasionally 
found  to  accompany  the  purulent  discharges  from  open 
wounds  is  usually  due  to  the  action  of  the  bacillus 
pyocyaneus.  According  to  recent  investigations  this 
bacillus  appears  to  be  very  widely  distributed. 

Morphology.  Slender  rods  from  0.3//  to  1/j.  broad  and 
from  2/^  to  6//  long;  frequently  united  in  pairs  or  in 
chains  of  four  to  six  elements;  occasionally  growing 
out  into  long  filaments  and  twisted  spirals.  The  bacil- 
lus is  actively  motile,  a  single  flagellum  being  attached 
to  one  end.  Does  not  form  spores.  Stains  with  the 
ordinary  aniline  colors;  does  not  stain  with  Gram's 
solution. 

Biological  Characters.  An  aerobic,  liquefying,  motile 
bacillus.  Capable  also  of  an  anaerobic  existence,  but 
then  produces  no  pigment.  Grows  readily  on  all  arti- 
ficial culture  media  at  the  room-temperature,  though 
best  at  37°  C.,  and  gives  to  some  of  them  a  bright 
green  color  in  the  presence  of  oxygen.  In  gelatin 
plate  cultures  the  colonies  are  rapidly  developed, 
imparting  to  the  medium  a  fluorescent  green  color; 
liquefaction  begins  at  the  end  of  two  or  three  days, 


536  BACTERIOLOGY. 

and  by  the  fifth  day  the  gelatin  is  usually  all  lique- 
fied. The  deep  colonies,  before  liquefaction  sets  in, 
appear  as  round,  granular  masses  with  scalloped  mar- 
gins, having  a  yellowish-green  color;  the  surface  col- 
onies have  a  darker  green  centre,  surrounded  by  a 
delicate,  radiating  zone.  In  stick  cultures  in  gelatin 
liquefaction  occurs  at  first  near  the  surface,  in  the  form 
of  a  small  funnel,  and  gradually  extends  downward; 
later  the  liquefied  gelatin  is  separated  from  the  solid 
part  of  the  medium  by  a  horizontal  plane,  a  greenish- 
yellow  color  being  imparted  to  that  portion  which  is 
in  contact  with  the  air.  On  agar  a  wrinkled,  moist, 
greenish-white  layer  is  developed,  while  the  surround- 
ing medium  is  bright  green;  this  subsequently  becomes 
darker  in  color,  changing  to  blue-green  or  almost  black. 
In  bouillon  the  green  color  is  produced,  and  the  growth 
appears  as  a  delicate,  flocculent  sediment.  Milk  is  coag- 
ulated with  coincident  acid  reaction. 

There  is  some  difference  of  opinion  with  regard  to 
the  pigments  produced  by  the  bacillus  pyocyaneus. 
Gessard's  view  is  that  two  pigments  are  produced  by 
this  bacillus — one  of  a  fluorescent  green  and  the  other 
(pyocyanin)  of  a  blue  color.  Pyocyanin  is  soluble  in 
chloroform,  and  may  be  obtained  from  pure  solution 
in  long,  blue  needles.  This  pigment,  which  is  thus 
extracted  by  chloroform,  distinguishes  the  bacillus 
pyocyaneus  from  other  fluorescing  bacteria. 

Pathogenesis.  This  bacillus  is  very  widely  distributed 
in  nature;  it  is  found  on  the  healthy  skin  of  man, 
in  purulent  discharges  and  in  serous  wound  secre- 
tions. Its  presence  in  wounds  greatly  delays  the 
process  of  repair  and  may  give  rise  to  a  general 
depression  of  the  vital  powers  from  the  absorption  of 


BACILLUS  PYOCTANEUS.  537 

its  toxic  products.  Its  pathogenic  effects  on  animals 
have  been  carefully  studied.  It  is  pathogenic  for 
guinea-pigs  and  rabbits.  Subcutaneous  or  intra-peri- 
toneal  injections  of  not  too  small  quantities  of  a 
recent  culture — 1  c.c.  or  more  of  a  bouillon  culture — 
usually  cause  the  death  of  the  animal  in  from  twenty- 
four  to  thirty-six  hours.  Subcutaneous  inoculations  pro- 
duce an  extensive  inflammatory  oedema  and  purulent 
infiltration  of  the  tissues;  a  serofibrinous  or  purulent 
peritonitis  is  induced  by  the  introduction  of  the  bacillus 
into  the  peritoneal  cavity.  The  bacilli  multipy  in  the 
body,  and  may  be  found  in  the  serous  or  purulent  fluid 
in  the  subcutaneous  tissues  or  abdominal  cavity  as  well 
as  in  the  blood  and  various  organs.  When  smaller 
quantities  are  injected  subcutaneously  the  animal  usu- 
ally recovers,  only  a  local  inflammatory  reaction  being 
set  up  (abscess),  and  it  is  subsequently  immune  against 
a  second  inoculation  with  doses  which  would  prove 
fatal  to  an  unprotected  animal.  Immunity  may  also 
be  secured  by  the  injection  of  a  considerable  amount 
of  a  sterilized  culture.  It  is  interesting  to  note  that 
Bouchard,  Charrin,  and  Guignard  have  shown  that  in 
rabbits  which  have  been  inoculated  with  a  culture  of 
the  bacillus  anthracis  a  fatal  result  may  be  prevented 
by  inoculating  the  same  animal  soon  after  with  a  pure 
culture  of  the  bacillus  pyocyaneus.  Similar  results 
have  been  obtained  by  Wood  head  and  Wood  by  the 
injection  of  sterilized  cultures  of  this  bacillus,  made 
immediately  after  injection  with  the  anthrax  bacillus. 
Loew  and  Emmerich  have  shown  that  the  enzymes 
produced  in  the  pyocyaneus  cultures  are  capable  of  de- 
stroying many  forms  of  bacteria  in  the  test-tube,  and 
have  slight  protecting  value  in  the  body. 


538  BACTERIOLOGY. 

Our  knowledge  of  the  pathogenic  importance  of  the 
bacillus  pyocyaneus  in  human  diseases  has  been  much 
increased  by  recent  investigations.  Thus  cases  have 
been  reported  in  which  this  bacillus  has  been  obtained 
in  pure  culture  from  pus  derived  from  the  tympanic 
cavity  in  disease  of  the  middle  ear,  from  cases  of  oph- 
thalmia and  bronchopneumonia.  Kruse  and  Pasquale 
have  found  the  same  micro-organism  in  three  cases  of 
idiopathic  abscess  of  the  liver,  in  two  of  them  in  immense 
numbers  and  in  pure  culture.  Ernst  and  Schiirmayer 
report  the  presence  of  the  bacillus  pyocyaneus  in  serous 
inflammation  of  the  pericardial  sac  and  of  the  knee- 
joint.  Ehlers  gives  the  history  of  a  disease  in  two 
sisters  who  were  attacked  simultaneously  with  fever, 
albuminuria,  and  paralysis.  It  was  thought  that  they 
would  turn  out  to  be  typhoid  fever  or  meningitis,  but 
on  the  twelfth  day  there  was  an  eruption  of  blisters, 
from  the  contents  of  which  the  bacillus  pyocyaneus  was 
isolated.  Jadkewitsch  reports  the  case  of  a  patient 
suffering  from  eczema  of  the  lower  extremities,  in 
whom  three  times  during  a  period  of  ten  years  there 
was  eruption  of  boils  containing  blue  pus,  with  accom- 
panying symptoms  of  poisoning,  emaciation,  prostra- 
tion, diarrhoea,  and  paresis.  Krambals  refers  to  seven 
cases  in  which  a  general  pyocyaneous  infection  occurred, 
and  adds  an  eighth  from  his  own  experience.  In  this 
the  bacillus  pyocyaneus  was  obtained  post-mortem  from 
green  pus  in  the  pleural  cavity,  from  serum  in  the  peri- 
cardial sac,  and  from  the  spleen  in  pure  culture. 
Schimmelbush  states  that  a  physician  injected  0.5  c.c. 
of  sterilized  (by  heat)  culture  into  his  forearm.  As  a 
result  of  this  injection,  after  a  few  hours  he  had  a 
slight  chill,  followed  by  fever,  which  at  the  end  of 


BACILLUS  PROTEUS  VULGARIS.  539 

twelve  hours  reached  38.8°  C.;  an  erysipelatous-like 
swelling  of  the  forearm  occurred,  and  the  glands  in 
the  axilla  were  swollen  and  painful.  Neumann  has 
obtained  the  bacillus  pyocyaneus  in  pure  culture  in 
two  cases  of  hsematemesis  and  melsena  of  the  new-born 
from  the  blood  and  other  organs.  Lartigau  found  it 
in  well-water,  and  in  great  abundance  in  the  intestinal 
discharges  of  a  number  of  cases  made  ill  by  drinking 
the  water. 

We  may,  therefore,  conclude  from  these  facts  that 
the  bacillus  pyocyaneus,  although  ordinarily  non-patho- 
genic for  man,  may  under  certain  conditions  become  a 
dangerous  source  of  infection.  Children  would  seem 
to  be  particularly  susceptible  to  this  infection. 

The  differential  diagnosis  of  the  pyocyaneus  from 
other  fluorescing  bacteria  is  easy  enough  as  long  as  it 
retains  its  pigment-producing  property.  When  an  agar 
culture  is  agitated  with  chloroform  a  blue  coloration 
demonstrates  the  presence  of  this  bacillus.  When  the 
pyocyanin  is  no  longer  formed,  however,  the  diagnosis 
is  by  no  means  easy,  particularly  when  the  pathogenic 
properties  are  also  gone. 

BACILLUS  PROTEUS   VULGAEIS. 

This  bacillus,  which  is  one  of  the  most  common  and 
widely  distributed  putrefactive  bacteria,  was  discovered 
by  Hauser  (1885)  along  with  other  species  of  proteus 
in  putrefying  substances.  These  bacteria  were  formerly 
included  under  the  namea  bacterium  termo  "  by  previous 
observers,  who  applied  this  name  to  any  minute  motile 
bacilli  found  in  putrefying  infusions. 

Morphology.  Bacilli  varying  greatly  in  size;  most 
commonly  occurring  0.6//  broad  and  1.2/*  long,  but 


540  BACTERIOLOGY. 

shorter  and  longer  forms  may  also  be  seen,  even  grow- 
ing out  into  flexible  filaments,  which  are  sometimes 
more  or  less  wavy  or  twisted  like  braids  of  hair.  The 
bacillus  does  not  form  spores,  and  stains  readily  with 
f  uchsin  or  gentian-violet. 

Biological  Characters.  An  aerobic,  facultative  anaer- 
obic, liquefying,  motile  bacillus.  Grows  rapidly  in 
the  usual  culture  media  at  the  room-temperature. 

Growth  on  Gelatin.  The  growth  upon  gelatin  plates 
containing  5  per  cent,  of  gelatin  is  very  characteristic. 
At  the  end  of  ten  or  twelve  hours  at  room-tem- 
perature small  round  depressions  in  the  gelatin  are 
observed,  which  contain  liquefied  gelatin  and  a  whitish 
mass  consisting  of  bacilli  in  the  centre.  Under  a  low- 
power  lens  these  depressions  are  seen  to  be  surrounded 
by  a  radiating  zone  composed  of  two  or  more  layers, 
outside  of  which  is  a  zone  of  a  single  layer,  from  which 
amoeba-like  processes  extend  upon  the  surface  of  the 
gelatin.  These  processes  are  constantly  undergoing 
changes  in  their  form  and  position.  The  young  colo- 
nies deep  down  in  the  gelatin  are  somewhat  more 
compact,  and  rounded  or  humpbacked;  later  they 
are  covered  with  soft  down;  then  they  form  irregular, 
radiating  masses  and  simulate  the  superficial  colonies. 
But  it  is  difficult  to  describe  all  the  forms  which  the 
proteus  vulgaris  takes  on  in  all  the  stages  of  its  growth 
on  gelatin  plates.  When  the  consistency  of  the  medium 
is  more  solid,  as  in  10  per  cent,  gelatin,  the  liquefac- 
tion and  migration  of  surface  colonies  are  more  or  less 
retarded.  In  gelatin  stick  cultures  the  growth  is  less 
characteristic — liquefaction  takes  place  rapidly  along 
the  line  of  puncture,  and  soon  the  entire  contents  of 
the  tube  are  liquefied. 


BACILLUS  PROTEUS  VULGARIS.  541 

Upon  nutrient  agar  a  rapidly  spreading,  moist,  thin, 
grayish-white  layer  appears,  and  migration  of  the  col- 
onies also  occurs.  Milk  is  coagulated,  with  the  pro- 
duction of  acid. 

The  cultures  in  media  containing  albumin  or  gelatin 
have  a  disagreeable,  putrefactive  odor,  and  become  alka- 
line in  reaction.  Growth  is  most  luxuriant  at  a  tem- 
perature of  24°  C.,  but  is  plentiful  also  at  37°  C.  It 
is  a  facultative  anaerobe  and  grows  also  in  the  presence 
of  oxygen,  but  the  proteus  then  loses  its  power  of  lique- 
fying gelatin.  It  produces  indol  and  phenol  from  pep- 
tone solutions.  The  proteus  develops  fairly  well  in 
urine,  and  decomposes  urea  into  carbonate  of  ammonia. 

Pathogenesis.  This  bacillus  is  pathogenic  for  rabbits 
and  guinea-pigs  when  injected  in  large  quantities  into 
the  circulation,  into  the  abdominal  cavity,  or  subcuta- 
neously,  producing  death  of  the  animals  with  symptoms 
of  poisoning.  Hauser  has  obtained  the  bacillus  proteus 
vulgaris  from  a  case  of  purulent  peritonitis,  from  puru- 
lent puerperal  endometritis,  and  from  a  phlegmonous 
inflammation  of  the  hand.  Brunner  also  reports  simi- 
lar infections  in  which  this  organism  was  found  asso- 
ciated with  pus  cocci,  and  Charrin  describes  a  case  of 
pleuritis  during  pregnancy  in  which  the  proteus  was 
present  and  a  foul-smelling  secretion  was  produced. 
Death  in  this  case,  which  ensued  without  further  com- 
plication, is  said  to  have  been  due  probably  to  the 
poisonous  products  of  the  proteus. 

An  interesting  example  of  pure  toxaemia  resulting 
from  the  toxin  of  the  proteus  is  reported  by  Levy : 
While  conducting  some  experiments  on  this  organism 
he  had  an  opportunity  of  making  a  bacteriological  ex- 
amination in  the  case  of  a  man  who  died  after  a  short 


542  BACTERIOLOGY. 

attack  of  cholera  morbus.  From  the  vomited  material 
and  the  stools  he  obtained  a  pure  culture  of  the  pro- 
teus;  but  the  blood,  collected  at  the  autopsy,  was  sterile. 
In  the  meantime  seventeen  other  persons  who  had  eaten 
at  the  same  restaurant  were  taken  sick  in  the  same 
way.  Upon  examination  at  the  restaurant  it  was  found 
that  the  bottom  of  the  ice-chest  in  which  the  meat  was 
kept  was  covered  with  a  slimy,  brown  layer,  which 
gave  off  a  disagreeable  odor.  Cultures  from  this  gave 
the  proteus  as  the  principal  organism  present.  Injec- 
tions into  animals  of  the  pure  cultures  produced  similar 
symptoms  as  occurred  in  the  human  subjects.  Levy 
concludes  that  in  so-called  "  flesh-poisoning  "  bacteria 
of  this  group  are  chiefly  concerned,  and  that  the  patho- 
genic effects  are  due  to  toxic  products  evolved  during 
their  development. 

Booker,  from  his  extended  researches  into  this  sub- 
ject, concludes  that  the  proteus  plays  an  important  part 
in  the  production  of  the  morbid  symptoms  which  char- 
acterize cholera  infantuna.  Proteus  vulgaris  was  found 
in  the  al vine  discharge  in  a  large  proportion  of  the  cases 
examined  by  him,  but  was  not  found  in  the  feces  of 
healthy  infants.  "The  prominent  symptoms  in  the 
cases  of  cholera  infantum  in  which  the  proteus  bacteria 
were  found  were  drowsiness,  stupor,  emaciation,  and 
great  reduction  in  flesh,  more  or  less  collapse,  frequent 
vomiting  and  purging,  with  watery  and  generally  offen- 
sive stools." 

Next  to  the  bacillus  coli  communis  the  proteus  vul- 
garis appears  to  be  the  micro-organism  most  frequently 
concerned  in  the  etiology  of  pyelonephritis.  In  cases 
of  cystitis  and  of  pyelonephritis  this  bacillus  is  often 
found  in  pure  cultures  or  associated  with  other  bac- 
teria. It  probably  gets  into  the  bladder  chiefly  through 


BACILLUS  OF  MALIGNANT  (EDEMA.         543 

catheterization.  From  the  animal  experiments  of  the 
authors  above  mentioned,  simple  injection  of  pure  cul- 
tures of  proteus  into  the  bladder,  without  artificial  sup- 
pression of  urine,  invariably  produces  severe  cystitis. 
The  fact  that  this  organism  grows  in  urine  is  sufficient 
to  account  for  the  extension  of  the  purulent  process 
finally  to  the  kidneys. 

The  proteus  vulgaris  is,  however,  a  harmles  parasite 
when  located  in  the  mucous  membrane  of  the  nasal 
cavities.  Here  it  only  decomposes  the  secretions,  with 
the  production  of  a  putrefactive  odor.  On  the  whole, 
considering  the  very  wide  distribution  of  this  organism 
in  nature,  it  is  remarkable  how  few  diseases  are  pro- 
duced by  it. 

BACILLUS   OF   MALIGNANT   (EDEMA. 

This  bacillus  is  widely  distributed,  being  found  in 
the  superficial  layers  of  the  soil,  in  putrefying  sub- 
stances, in  the  blood  of  animals  which  have  been  suffo- 
cated (by  invasion  from  the  intestine),  in  foul  water, 
etc.  It  was  discovered  (1877)  by  Pasteur  in  animals 
after  injections  of  putrefying  liquids,  and  named  by 
him  "  vibrion  septique."  He  recognized  its  anaerobic 
nature,  but  did  not  obtain  it  in  pure  culture.  Koch 
(1881)  carefully  studied  this  micro-organism,  described 
it  in  detail,  and  gave  it  the  name  "  bacillus  cedematous 
maligni."  It  was  isolated  first  in  pure  culture  by 
Liborius. 

Morphology.  The  oedema  bacillus  is  a  rod  of  from 
0.8/2  to  \IJL  in  width,  and  of  very  varying  length,  from 
2//  to  10//  or  more,  according  to  .the  conditions  of  its 
cultivation  and  growth.  It  is  usually  found  in  pairs, 
joined  end  to  end,  but  may  occur  in  chains  or  long 
filaments.  It  forms  spores,  and  these  are  situated 


544  BACTERIOLOGY. 

in  or  near  the  middle  of  the  body  of  the  rods.  The 
spores  vary  in  length  and  are  oval  in  form,  being  often 
of  greater  diameter  than  the  bacilli,  to  which  they  give 
a  more  or  less  oval  or  spindle  shape. 

The  bacilli  stain  readily  by  the  usual  aniline  colors 
employed,  but  are  decolorized  by  Gram's  method. 

Biological  Characters.  A  strictly  anaerobic,  liquefy- 
ing, motile  bacillus.  Forms  spores.  It  grows,  however, 
in  all  the  usual  culture  media  in  the  absence  of  oxygen. 
Development  takes  place  at  the  room-temperature,  but 
more  rapidly  and  abundantly  at  37°  C. 

Growth  in  Gelatin.  This  bacillus  may  be  cultivated 
in  ordinary  nutrient  gelatin,  but  the  growth  is  more 
abundant  in  glucose-gelatin  containing  1  or  2  per  cent, 
of  glucose.  Gas-bubbles  are  formed  and  the  gelatin 
liquefies. 

Growth  on  Agar.  On  agar  plates  the  colonies  appear 
as  dull,  whitish  points,  irregular  in  outline,  and  when 
examined  under  a  low-power  lens  are  seen  to  be  com- 
posed of  a  dense  network  of  interlacing  threads  radi- 
ating irregularly  from  the  centre  toward  the  periphery. 

Blood-serum  is  rapidly  liquefied,  with  the  production 
of  gas.  Cultures  of  the  malignant  oedema  bacillus  give 
off  a  peculiar,  disagreeable  odor. 

Pathogenesis.  The  bacillus  of  malignant  oedema  is 
especially  pathogenic  for  mice,  guinea-pigs,  and  rabbits, 
although  man,  horses,  dogs,  goats,  sheep,  calves,  pigs, 
chickens,  and  pigeons  are  also  susceptible.  A  small 
quantity  of  a  pure  culture  injected  beneath  the  skin  of 
a  susceptible  animal  gives  rise  to  an  extensive  hemor- 
rhagic  oedema  of  the  subcutaneous  connective  tissue, 
which  extends  over  the  entire  surface  of  the  abdomen 
and  thorax,  causing  hypersemia  and  redness  of  the 
superficial  muscles.  There  is  no  odor  developed,  and 


BACILLUS  AEROGENES  CAPSULATUS.       545 

little,  if  any,  production  of  gas.  In  infection  with 
garden  earth,  owing  to  the  presence  of  associated  ba- 
cilli, the  effused  serum  is  frothy  from  the  development 
of  gas,  and  possesses  a  putrefactive  odor.  The  disease, 
in  natural  infection  caused  by  the  contamination  of 
wounds  with  earth  or  feces,  runs  the  course  above  de- 
scribed. Simple  abrasion  of  the  skin  is  not  sufficient 
to  produce  infection;  owing  to  the  bacillus  being  capable 
only  of  an  anaerobic  existence,  the  poison  must  pene- 
trate deep  into  the  tissues.  Malignant  oedema  is  con- 
fined mostly  to  the  domestic  animals,  but  cases  have 
also  been  reported  in  man. 

Animals  which  recover  from  malignant  oedema  are 
subsequently  immune.  Artificial  immunity  may  be 
induced  in  guinea-pigs  by  injecting  filtered  cultures  of 
the  malignant  oedema  bacillus  in  harmless  quantities. 

BACILLUS  AEROGENES  OAPSULATUS. 

Found  by  Welch  in  the  bloodvessels  of  a  patient 
suffering  with  aortic  aneurism;  on  autopsy,  made  in 
cool  weather  eight  hours  after  death,  the  vessels  were 
observed  to  be  full  of  gas-bubbles.  Since  then  it  has 
been  found  in  a  number  of  cases  in  which  gas  has  de- 
veloped from  within  sixty  hours  of  death  until  some 
hours  after  death.  These  cases  are,  as  a  rule,  marked  by 
delirium,  rapid  pulse,  high  temperature,  and  the  devel- 
opment of  emphysema  and  discoloration  of  the  diseased 
area,  or  of  marked  abdominal  distention  when  the  peri- 
toneal cavity  is  involved. 

Morphology.  Straight  or  slightly  curved  rods,  with 
rounded  or  sometimes  square-cut  ends;  somewhat 
thicker  than  the  anthrax  bacilli  and  varying  in  length ; 
occasionally  long  threads  and  chains  are  seen.  The 

35 


546  BACTERIOLOGY. 

bacilli  in  the  animal  body,  and  sometimes  in  cultures, 
are  enclosed  in  a  transparent  capsule. 

Biological  Characters.  An  anaerobic,  non-motile,  non- 
liquefying  bacillus.  Does  not  form  spores.  Grows  at 
the  room-temperature,  but  more  rapidly  at  37°  C.,  in 
the  usual  culture  media  in  the  absence  of  oxygen,  and 
is  accompanied  by  the  production  of  gas.  Nutrient 
gelatin  is  not  liquefied  by  the  growth  of  this  bacillus, 
but  it  is  gradually  peptonized.  In  agar  colonies  are 
developed  which  are  from  1  to  2  mm.  or  more  in 
diameter,  grayish-white  in  color,  and  in  the  form  of 
flattened  spheres,  ovals,  or  irregular  masses,  beset  with 
hair-like  projections.  Bouillon  is  diffusely  clouded,  and 
a  white  sediment  is  formed.  Milk  is  rapidly  coagulated. 

Pathogenesis.  Usually  non-pathogenic  in  healthy 
animals,  although  Dunham  found  that  the  bacillus  taken 
freshly  from  human  infection  is  sometimes  very  virulent. 
When  quantities  up  to  2.5  c.c.  of  fresh  bouillon  cultures 
are  injected  into  the  circulation  of  rabbits  and  the  ani- 
mals killed  shortly  after  the  injection,  the  bacilli  de- 
velop rapidly,  with  an  abundant  formation  of  gas  in 
the  bloodvessels  and  organs,  especially  the  liver.  The 
following  is  one  of  the  best  methods  of  obtaining 
the  bacilli :  The  material  suspected  to  contain  the  ba- 
cillus alone  or  associated  with  other  bacteria  is  injected 
into  rabbits,  which  are  killed,  kept  at  37°  C.,  and  cultures 
made  twenty-four  hours  later  from  their  bodies. 

It  is  suggested  by  Welch  that  in  some  of  the  cases  in 
which  death  has  been  attributed  to  the  entrance  of  air 
into  the  veins  the  gas  found  at  the  autopsy  may  not 
have  been  atmospheric  air,  but  may  have  been  produced 
by  this  or  some  similar  micro-organism  entering  the  cir- 
culation and  developing  shortly  before  and  after  death. 
The  bacillus  has  been  found  in  the  dust  of  hospitals. 


CHAPTER   XXXII. 

BACILLUS  ANTHRACIS — BACILLUS  ANTHRACIS  SYMP- 
TOMATICI  (ANTHRAX  BACILLUS). 

BACILLUS  ANTHEACIS. 

ANTHRAX  is  an  acute  infectious  disease  which  is 
very  prevalent  among  animals,  particularly  sheep  and 
cattle.  Geographically  and  zoologically  it  is  the  most 
wide-spread  of  all  infectious  disorders.  It  is  much 
more  common  in  Europe  and  in  Asia  than  in  America. 
The  ravages  among  herds  of  cattle  in  Russia  and  Sibe- 
ria, and  among  sheep  in  certain  parts  of  France,  Hun- 
gary, Germany,  Persia,  and  India,  are  not  equalled  by 
any  other  animal  plague.  Local  epidemics  have  occa- 
sionally occurred  in  England,  where  it  is  known  as 
splenic  fever.  In  this  country  the  disease  is  rare.  In 
infected  districts  the  greatest  losses  are  incurred  during 
the  hot  months  of  summer. 

The  disease  also  occurs  in  man  as  the  result  of  in- 
fection, either  through  the  skin,  the  intestines,  or  in 
rare  instances  through  the  lungs.  It  is  found  in  per- 
sons whose  occupations  bring  them  into  contact  with 
animals  or  animal  products,  as  stablemen,  shepherds, 
tanners,  butchers,  and  those  who  work  in  wool  and 
hair.  Two  forms  of  the  disease  have  been  described 
— the  external  anthrax,  or  malignant  pustule,  and  the 
internal  anthrax,  of  which  there  are  intestinal  and 


548  BACTERIOLOGY. 

pulmonary  forms,  the  latter  being  known  as  "  wool- 
sorter's  disease." 

Owing  to  the  fact  that  anthrax  was  the  first  infec- 
tious disease  which  was  shown  to  be  caused  by  a  specific 
micro-organism,  and  to  the  close  study  which  it  received 
in  consequence,  this  disease  has  probably  contributed 
more  to  our  general  knowledge  of  bacteriology  than 
any  other  infectious  malady. 

Pollender  observed  in  1849  that  the  blood  of  animals 
suffering  from  splenic  fever  always  contained  minute 
rod-shaped  bacteria.  Davaine,  in  1863,  announced  to 
the  French  Academy  of  Sciences  the  results  of  his  in- 
oculation experiments,  and  asserted  the  etiological  rela- 
tion of  the  micro-organism  to  the  disease  with  which 
his  investigation  showed  it  to  be  constantly  associated. 
For  a  long  time  this  conclusion  was  energetically  op- 
posed until,  in  1879,  Pasteur,  Koch  and  others  estab- 
lished its  truth  by  obtaining  the  bacillus  in  pure  cultures 
and  showing  that  the  inoculation  of  these  cultures  pro- 
duced anthrax  in  susceptible  animals  as  certainly  as  did 
the  blood  of  an  animal  recently  dead  from  the  disease. 

Morphology,  Slender,  cylindrical,  non-motile  rods, 
having  a  breadth  of  \p  to  1.25//,  and  ranging  from  2 
or  3//  to  20  or  25//  in  length.  They  vary  thus  very 
much  in  their  length.  Sometimes  short,  isolated  rods 
are  seen,  and,  again,  shorter  or  longer  chains  or  threads 
made  up  of  several  rods  joined  end  to  end.  In  suitable 
culture  media  very  long,  flexible  filaments  may  be 
observed,  which  are  frequently  united  in  twisted  or 
plaited,  cord-like  bundles.  (See  Fig.  71  and  Fig.  13, 
p.  47,  and  Fig.  17,  p.  207.)  These  filaments  in  hang- 
ing drop  cultures,  before  the  development  of  spores, 
appear  to  be  homogeneous  or  nearly  so;  but  in  stained 


BACILLUS  ANTHRACIS. 


549 


preparations  they  are  seen  to  be  composed  of  a  series  of 
rectangular,  deeply  stained  segments.  When  obtained 
directly  from  the  blood  of  an  infected  animal  the  free 
ends  of  the  rods  are  slightly  rounded,  but  those  com- 
ing in  contact  with  one  another  are  quite  square.  In 
cultures  the  ends  are  seen  to  be  a  trifle  thicker  than 
the  body  of  the  cell  and  somewhat  concave,  giving  the 
appearance  of  joints  of  bamboo.  At  one  time  much  stress 

FIG.  71. 


Anthrax  bacillus.,   X  900  diameters.    Agar  culture. 

was  laid  upon  these  peculiarities  as  distinguishing  marks 
of  the  anthrax  bacillus;  but  it  has  been  found  that  these 
are  the  effects  of  artificial  cultivation  and  not  necessarily 
characteristic  of  the  organism  under  all  conditions.  An- 
other peculiarity  of  this  bacillus  is  that  it  is  enclosed 
in  a  transparent  envelope  or  capsule,  which  in  stained 
preparations  may  be  distinguished  by  its  taking  on  a 
lighter  stain  than  the  deeply  stained  rods  which  it  sur- 
rounds. 


550 


BACTERIOLOGY. 


Under  favorable  conditions  in  cultures  spores  are 
developed  in  the  bacilli.  These  spores  are  elliptical 
in  shape  and  about  one  and  a  half  times  longer  than 
broad.  They  first  appear  as  small,  refractive  gran- 
ules distributed  at  regular  intervals,  one  in  each  rod. 
As  the  spore  develops  the  mother-cell  becomes  less  and 
less  distinct,  until  it  disappears  altogether,  the  com- 
plete oval  spore  being  set  free  by  its  dissolution.  (Fig. 
72,  Fig.  13,  p.  47,  and  Fig.  17,  p.  207).  Irregular 
sporulation  sometimes  takes  place,  and  occasionally 
there  is  no  spore-formation,  as  in  varieties  of  non-spore 
bearing  anthrax. 


FIG.  72. 


Spores  heavily  stained  (in  specimen  red).    Bodies  of  disintegrating  bacilli 
faintly  stained  (in  specimen  blue).    X  1000  diameters. 

The  anthrax  bacillus  stains  readily  with  all  the  aniline 
colors,  and  also  by  Gram's  method,  when  not  left  too 
long  in  the  decolorizing  iodine  solution.  In  sections 
good  results  may  be  obtained  by  the  employment  of 
Gram's  solution  in  combination  with  carmine,  but  when 


BACILLUS  ANTHRACIS.  551 

only  a  few  bacilli  are  present  this  method  is  not  always 
reliable,  as  some  of  the  bacilli  are  generally  decolorized. 

Biological  Characters.  The  anthrax  bacillus  grows 
easily  in  a  variety  of  nutrient  media  at  a  temperature 
from  18°  to  43°  C.,  37°  C.  being  the  most  favorable 
temperature.  Under  12°  C.  no  development  takes 
place,  as  a  rule,  though  by  gradually  accustoming  the 
bacillus  to  a  lower  temperature  it  may  be  induced  to 
grow  under  these  conditions.  Under  14°  C.  and  above 
43°  C.  spore-formation  ceases.  The  lower  limit  of 
growth  and  sporulation  is  of  practical  significance  in 
determining  the  question  whether  development  can 
occur  in  •  the  bodies  of  animals  dead  from  anthrax 
when  buried  at  certain  depths  in  the  earth.  Kitasato 
has  shown  that  at  a  depth  of  1.5  metres  the  earth  in 
July  has  a  temperature  of  15°  C.  at  most,  and  that 
under  these  conditions  a  scanty  sporulation  of  anthrax 
bacilli  is  possible,  but  that  at  a  depth  of  2  metres  sporu- 
lation no  longer  occurs.  The  anthrax  bacillus  is  aerobic 
— that  is,  its  growth  is  considerably  enhanced  by  the 
presence  of  oxygen — but  it  grows  also  under  anaerobic 
conditions,  as  is  shown  by  its  growth  at  the  bottom  of 
the  line  of  puncture  in  stick  cultures  in  solid  media; 
but  under  these  conditions  it  no  longer  produces  the 
peptonizing  ferment  which  it  does  with  free  access  of 
air.  Furthermore,  the  presence  of  oxygen  is  absolutely 
necessary  for  the  formation  of  spores,  while  carbonic 
acid  gas  retards  sporulation.  This  explains,  perhaps, 
why  sporulation  does  not  take  place  within  the  animal 
body  either  before  or  after  death. 

This  bacillus  grows  best  in  neutral  or  slightly  alka- 
line media.  It  may  be  cultivated  in  infusions  of  meat 
or  of  various  vegetables,  in  urine;  etc,,  provided  the 


552 


BACTERIOLOGY. 


reaction  be  not  decidedly  acid,  which  arrests  develop- 
ment. It  grows  in  cow-dung  and  in  more  or  less 
contaminated  earth.  It  is  also  capable  of  leading  a 
saprophytic  existence.  The  bacillus  is  non-motile. 

Growth  in  Gelatin.  In  gelatin  plate  cultures,  at 
the  end  of  twenty-four  to  thirty-six  hours  at  24°  C., 
small,  white,  opaque  colonies  are  developed,  which 

FIG.  73. 


Colonies  of  bacillus  anthracis  upon  gelatin  plates,    a,  at  the  end  of  twenty- 
four  hours ;  6,  at  the  end  of  forty-eight  hours.     X  80.    (F.  FLtlGGE.) 

under  a  low-power  lens  are  seen  to  be  dark  gray  in 
the  centre  and  surrounded  by  a  greenish,  irregular 
border,  made  up  of  wavy  filaments.  As  the  colony 
develops  on  the  surface  of  the  gelatin  these  wavy 
filaments  spread  out,  until  finally  the  entire  colony 
consists  of  a  light  gray,  tangled  mass,  which  has  been 
likened  to  a  Medusa  head  (Fig.  73). 

At  the  same  time  the  gelatin  begins  to  liquefy,  and 
the  colony  is  soon  surrounded  by  the  liquefied  medium, 


BA  GILL  US  ANTHRA  CIS.  553 

upon  the  surface  of  which  it  floats  as  an  irregular,  white 
pellicle.  In  gelatin  stick  cultures  at  first  development 
occurs  along  the  line  of  puncture  as  a  delicate  white 
thread,  from  which  irregular,  hair-like  projections  soon 
extend  perpendicularly  into  the  culture  medium,  the 
growth  being  most  luxuriant  near  the  surface,  but  con- 
tinuing also  below.  At  the  end  of  two  or  three  days 
liquefaction  of  the  medium  commences  at  the  surface 
and  gradually  progresses  downward. 

Growth  on  Agar.  The  growth  on  agar  plate  cul- 
tures in  the  incubator  at  37°  C.  is  similar  to  that  on 
gelatin,  and  is  still  more  characteristic  and  beautiful  in 
appearance.  A  grayish-white  layer  is  formed  on  the 
surface  within  twenty-four  hours,  which  spreads  rapidly 
and  is  seen  to  be  made  up  of  interlaced  threads. 

In  bouillon  the  growth  is  characterized  by  the  forma- 
tion of  flaky  masses,  which  sink  as  a  sediment  to  the 
bottom  of  the  tube,  leaving  the  supernatant  liquid  clear. 

Spore  formation,  as  already  noted,  only  takes  place 
in  the  free  presence  of  oxygen,  and  at  a  temperature  of 
15°  to  43°  C.  There  is  no  development  of  spores  at  a 
greater  depth  than  1.5  metres  in  the  earth,  or  in  the 
bodies  of  living  or  dead  animals;  but  spores  may  be 
found  in  the  fluids  containing  the  bacilli  when  these 
come  in  contact  with  the  air,  as  in  bloody  discharges 
from  the  nostrils  or  from  the  bowels  of  the  dead  animal. 

There  are  certain  non- spore  bearing  species  of  an- 
thrax. Sporeless  varieties  have  also  been  produced 
artificially  by  cultivating  the  typical  anthrax  bacillus 
under  unfavorable  conditions.  The  addition  of  anti- 
septics, as  carbolic  acid,  favors  these  conditions.  Vari- 
eties differing  in  their  pathogenic  power  may  also  be 
produced  artificially.  Pasteur  produced  an  "  attenu- 


554  BACTERIOLOGY. 

ated  virus  "  by  keeping  his  cultures  for  a  considerable 
time  before  replanting  them  upon  fresh  soil.  Cham- 
berland  and  Roux  have  shown  that  cultivation  in  the 
presence  of  certain  chemical  substances  added  to  the 
culture  medium,  as  bichromate  of  potassium,  causes  an 
attenuation  of  virulence.  Attenuation  of  pathogenic 
power  is  also  effected  by  cultivation  in  the  body  of  a 
non-susceptible  animal,  like  the  frog  (Lubarsch,  Pe- 
truschky);  or  in  the  blood  of  a  rat  (Behring);  by  ex- 
posure to  sunlight  (Arloing);  to  heat,  50°  C.  for 
eighteen  minutes;  and  by  compressed  air  (Chauveau). 
'^Anthrax  cultures  containing  spores  retain  their  vital- 
ity for  years;  in  the  absence  of  spores  the  vitality  is 
much  more  rapidly  lost.  When  grown  in  liquids  rich 
in  albumin  the  bacilli  attain  a  considerable  degree  of 
resistance;  thus  dried  anthrax  blood  has  been  found  to 
retain  its  virulence  for  sixty  days,  while  dried  bouillon 
cultures  only  did  so  for  twenty-one  days.  Dried  anthrax 
spores  may  be  preserved  for  many  years  without  losing 
their  vitality  or  virulence.  They  also  resist  a  com- 
paratively high  temperature.  Exposed  in  dry  air  they 
require  a  temperature  of  140°  C.  maintained  for  three 
hours  to  destroy  them;  but  suspended  in  a  liquid  they 
are  destroyed  in  four  minutes  by  a  temperature  of 
100°  C.  The  bacilli,  in  the  absence  of  spores,  are  de- 
stroyed in  ten  minutes  by  a  temperature  of  54°  C. 
Anthrax  spores  in  a  desiccated  condition  are  destroyed 
in  four  hours  when  exposed  to  the  action  of  direct 
sunlight,  only  after  several  weeks  in  diffuse  daylight 
(Kruse). 

Pathogenesis.  The  anthrax  bacillus  is  pathogenic  for 
cattle,  sheep  (except  the  Algerian  race),  horses,  swine, 
mice,  guinea-pigs,  and  rabbits.  Bats,  cats,  dogs,  chick- 


BA  GILL  US  ANTHRA  CIS.  555 

ens,  owls,  pigeons,  and  frogs  are  but  little  susceptible 
to  infection.  Small  birds — the  sparrow  particularly — 
are  somewhat  susceptible.  Man,  though  subject  to 
local  infection  and  occasionally  to  internal  forms  of  the 
disease,  is  not  as  susceptible  as  some  of  the  lower  ani- 
mals. 

The  anthrax  bacillus  produces  in  susceptible  animals 
a  true  septicaemia.  Among  test  animals  mice  are  the 
most  susceptible,  succumbing  to  very  minute  injections 
of  a  slightly  virulent  virus;  next  guinea-pigs,  and  lastly 
rabbits,  both  of  these  animals  dying  after  inoculation 
with  virulent  bacilli.  Infection  is  most  promptly  pro- 
duced by  introduction  of  the  bacilli  into  the  circulation 
or  the  tissues,  but  inoculation  by  contact  with  wounds 
on  the  skin  also  cause  infection.  It  is  difficult  to  pro- 
dace  infection  by  the  ingestion  even  of  spores;  but  it 
may  readily  be  caused  by  inhalation,  particularly  by  the 
inhalation  of  spores. 

Subcutaneous  injections  of  these  susceptible  animals 
results  in  death  in  from  one  to  three  days.  Compara- 
tively little  local  reaction  occurs  immediately  at  the 
point  of  inoculation,  but  beyond  this  there  is  an  exten- 
sive oedema  of  the  tissues.  Very  few  bacilli  are  found 
in  the  blood,  but  in  the  internal  organs,  and  especially 
in  the  capillaries  of  the  liver,  the  kidneys,  and  the 
lungs,  they  are  present  in  great  numbers.  In  some 
places  the  capillaries  will  be  seen  to  be  stuffed  full  of 
bacilli,  as  in  the  glomeruli  of  the  kidneys,  and  hemor- 
rhages, probably  due  to  rupture  of  capillaries  by  the 
mechanical  pressure  of  the  bacilli  which  are  developing 
within  them,  may  occur.  The  pathological  lesions  in 
animals  infected  by  anthrax  are  not  marked  except  in 
the  spleen,  which,  as  in  other  forms  of  septicaemia,  is. 


556  BACTERIOLOGY. 

greatly  enlarged.  The  anthrax  bacilli  in  these  animals 
seem  to  live  almost  exclusively  in  the  bloodvessels  and 
to  leave  them  only  by  means  of  hemorrhages.  In  this 
way  they  reach — but  only  late  in  the  disease — -the 
various  secretions  of  the  body,  the  urine,  the  intestinal 
secretions,  and  occasionally  the  bile.  The  passage  of 
the  anthrax  bacillus  from  the  mother  to  the  foetus  in 
pregnant  females  is  possible,  as  has  been  shown  by  the 
investigations  of  Strauss,  Chamberlain,  and  others,  but 
it  very  rarely  occurs. 

Occurrence  in  Cattle  and  Sheep.  Cattle  and  sheep 
are  affected  chiefly  with  the  intestinal  form  of  an- 
thrax, infection  in  these  animals  commonly  resulting 
from  the  ingestion  of  food  containing  spores.  The 
bacillus  itself,  in  the  absence  of  spores,  is  quickly  de- 
stroyed by  the  gastric  juice  (Koch,  Gaffky,  Loffler). 
The  disease  usually  takes  a  rapid  course,  and  the  mor- 
tality is  high — 70  to  80  per  cent.  The  pathological 
lesions  consist  of  numerous  ecchymoses,  enlargement  of 
the  lymphatic  glands,  serous,  fatty,  and  hemorrhagic 
infiltration  of  the  mediastinum  and  mesentery,  of  the 
mucous  membranes  of  the  pharynx  and  larynx,  and 
particularly  of  the  duodenum,  great  enlargement  of  the 
spleen,  and  parenchymatous  changes  in  the  lymphatic 
organs.  The  blood  is  very  dark  and  tar-like.  Bacilli 
are  present  in  enormous  masses. 

^Sheep  are  also  subject  to  external  anthrax,  infection 
taking  place  by  way  of  the  skin;  cattle  are  seldom  in- 
fected in  this  way.  At  the  point  of  inoculation  there 
develops  a  hard,  circumscribed  boil — the  so-called  an- 
thrax carbuncle;  or  there  may  be  diffuse  oedema,  with 
great 'swelling  of  the  parts.  When  death  occurs  the 
appearances  are  similar  to  those  in  intestinal  anthrax, 


BA  CILL  US  AN  THE  A  CIS.  557 

except  that  the  duodenum  is  usually  less  affected;  but  in 
all  cases  metastasis  occurs  in  various  parts  of  the  body, 
brought  about,  no  doubt,  by  previous  hemorrhages. 

Occurrence  in  Man.  The  disease  does  not  occur 
spontaneously  in  man,  but  always  results  from  infec- 
tion, either  through  the  skin,  the  intestines,  or  occasion- 
ally by  inhalation  through  the  lungs.  It  is  usually 
produced  by  cutaneous  infection  through  inoculation  of 
exposed  surfaces — the  hands,  arms,  or  face.  Infection 
of  the  face  or  neck  would  seem  to  be  the  most  danger- 
ous, the  mortality  in  such  cases  being  26  per  cent.; 
while  infection  of  the  extremities  is  very  rarely  fatal — 
in  only  5  per  cent,  of  cases  (Nassarow  and  Miiller). 

External  anthrax  in  man  is  similar  to  this  form  of 
the  disease  in  animals.  There  are  two  forms  :  Malig- 
nant pustule  or  carbuncle,  and,  less  commonly,  malig- 
nant anthrax  oedema. 

In  malignant  pustule,  at  the  site  of  inoculations,  a  small 
papule  develops,  which  becomes  vesicular.  Inflamma- 
tory induration  extends  around  this,  and  within  thirty- 
six  hours  there  is  a  dark  brownish  eschar  in  the  centre, 
at  a  little  distance  from  which  there  may  be  a  series  of 
small  vesicles.  The  brawny  induration  may  be  extreme. 
There  may  also  be  considerable  oedema  of  the  parts.  In 
most  cases  there  is  no  fever;  or  the  temperature  at  first 
rises  rapidly  and  the  febrile  phenomena  are  marked. 
Death  may  take  place  in  from  three  to  five  days.  In 
cases  which  recover  the  symptoms  are  slighter.  In  the 
mildest  form  there  may  be  only  slight  swelling. 

Malignant  anthrax  cedema  occurs  in  the  eyelids,  and 
also  in  the  head  and  neck,  sometimes  the  hand  and  arm. 
It  is  characterized  by  the  absence  of  the  papule  and 
vesicle  forms,  and  by  the  most  extensive  oedema.  The 


558  BACTERIOLOGY. 

oedema  may  become  so  intense  that  gangrene  results; 
such  cases  usually  prove  fatal. 

The  bacilli  are  found  on  microscopical  examination 
of  the  fluid  from  the  pustule  shortly  after  infection; 
later  the  typical  anthrax  bacilli  are  often  replaced  by 
involution  forms.  In  this  case  resort  may  be  had  to 
cultures,  animal  inoculation,  or  examination  of  sections 
of  the  extirpated  tumor.  The  bacilli  are  not  present 
in  the  blood  until  just  before  death.  Along  with  the 
anthrax  bacilli  pus  cocci  are  often  found  in  the  pustule 
penetrating  into  the  dead  tissue. 

Internal  anthrax  is  much  less  common  in  man;  it 
does,  however,  occur  now  and  then.  There  are  two 
forms  of  this  :  the  intestinal  form,  or  mycosis  intesti- 
nalis,  and  the  pulmonic  form,  or  wool-sorter's  disease. 

Intestinal  anthrax  is  caused  by  infection  through  the 
stomach  and  intestines,  and  results  probably  from  the 
eating  of  raw  flesh  or  unboiled  milk  of  diseased  animals. 
That  the  eating  of  flesh  from  infected  animals  is  com- 
paratively harmless  is  shown  by  Gerlier,  who  states  that 
of  400  persons  who  were  known  to  have  eaten  such 
meat  not  one  was  affected  with  anthrax.  On  the  other 
hand,  an  epidemic  of  anthrax  was  produced  among  wild 
animals,  according  to  Jansen,  by  feeding  them  on  in- 
fected horse  flesh.  It  is  evident,  therefore,  that  there 
is  a  possibility  of  infection  being  caused  in  this  way. 
The  recorded  cases  of  intestinal  anthrax  in  man  have 
occurred  in  persons  who  were  in  the  habit  of  handling 
hides,  hair,  etc.,  which  were  contaminated  with  spores; 
in  those  who  were  conducting  laboratory  experiments, 
and  rarely  it  has  been  produced  by  the  ingestion  of  food, 
such  as  raw  ham  and  milk.  The  symptoms  produced 
in  this  disease  are  those  of  intense  poisoning — chill,  fol- 


BACILLUS  ANTHRACIS.  559 


lowed  by  vomiting,  diarrhoea,  moderate  fever,  and  pains 
in  the  legs  and  back.  In  acute  cases  there  may  be  dysp- 
noea, cyanosis,  and  toward  the  end  convulsions.  The 
pathological  lesions  are  similar  to  those  described  in 
animals. 

Wool-sorter's  disease,  or  pulmonic  anthrax,  is  found 
in  large  establishments  in  which  wool  and  hair  are  sorted 
and  cleansed,  and  is  caused  by  the  inhalation  of  dust  con- 
taminated with  anthrax  spores.  The  attack  comes  on 
with  chills,  prostration,  then  fever.  The  breathing  is 
rapid,  and  the  patient  complains  of  pain  in  the  chest. 
There  may  be  a  cough  and  signs  of  bronchitis.  The 
bronchial  symptoms  in  some  instances  are  pronounced. 
Death  may  occur  in  from  two  to  seven  days.  The  path- 
ological changes  produced  are  swelling  of  the  glands  of 
the  neck,  the  formation  of  foci  of  necrosis  in  the  air- 
passages,  oedema  of  the  lungs,  pleurisy,  bronchitis,  en- 
largement of  the  spleen,  and  parenchymatous  degener- 
ations. 

Many  theories  have  been  advanced  to  account  for  the 
occurrence  of  intestinal  anthrax  among  cattle  and  sheep, 
which  in  these  animals  is  the  most  wide-spread  form  of 
the  disease.  It  has  been  thought  that  infection  was 
produced  mainly  by  the  eating  of  food  contaminated  by 
anthrax  spores  derived  from  the  bodies  of  infected  ani- 
mals; but  only  in  rare  instances  has  it  been  possible  to 
trace  the  cause  of  the  disease  to  this  source.  The 
grazing  of  cattle  on  infected  pastures  has  also  been 
assigned  as  the  cause  of  the  disease;  but  this  does  not 
explain  the  occurrence  of  epidemics  or  the  infection  of 
cattle  on  pastures  which  have  never  before  been  visited 
by  animals  affected  with  anthrax.  By  some  anthrax 
has  been  called  a  miasmatic  disease  and  likened  to 


560  BACTERIOLOGY. 

malaria.  Occurring  as  it  does  at  the  same  season  of 
the  year — viz.,  from  June  to  October — and  being  con- 
nected apparently,  like  malaria,  in  some  way  with  the 
condition  of  the  soil,  there  is  a  certain  analogy  between 
these  two  diseases.  Anthrax  is  found  to  occur  mostly 
in  low,  swampy  localities,  where  the  soil  is  covered  with 
decaying  vegetable  matter,  and  subject  to  overflows  and 
freshets.  There  is  no  doubt  that  this  bacillus  is  able  to 
lead  a  saprophytic  existence  for  some  time,  under  favor- 
able conditions,  in  the  superficial  layers  of  the  soil,  re- 
maining latent  in  the  form  of  spores  and  retaining  its 
vitality;  but  why  an  epidemic  of  anthrax  occurs  one 
year  at  a  certain  place  and  at  the  same  place  the  next 
year  does  not,  it  is  not  easy  to  explain.  Pasteur  believes 
that  the  earth-worm  plays  an  important  part  in  bringing 
to  the  surface  and  distributing  the  spores  which  have 
been  propagated  in  the  buried  carcass  of  an  infected  ani- 
mal; but  Koch  has  shown  that  this  hypothesis  is  both 
improbable  and  superfluous.  Apart  from  the  fact  that 
spor ulation  does  not  normally  take  place  inside  the  bodies 
of  dead  animals,  the  earth-worm  is  ill  adapted  for  the 
transportation  of  anthrax  spores,  which  are  unfavorably 
affected  in  their  intestines.  Out  of  seventy-two  earth- 
worms examined  by  Ballinger  from  a  notoriously  infected 
locality,  only  one  contained  anthrax  spores.  Further- 
more, the  soil  in  places  where  such  carcasses  lie  buried 
is  already  saturated  with  the  fluids  and  other  products 
of  decomposition  of  the  body  of  the  dead  animal  contain- 
ing bacilli,  which  under  suitable  temperature  conditions 
may  form  spores  and  thus  infect  the  surface  of  the  land; 
though  it  is  possible  that  the  earth-worm,  in  some  in- 
stances, may  contribute  to  the  distribution  of  spores  to 
a  certain  extent.  It  would,  therefore,  seem  that  the  only 


BACILLUS  ANTHRACIS.  561 

plausible  explanation  which  can  be  offered,  in  the  light  of 
our  present  knowledge,  for  the  solution  of  thib  problem, 
is  the  supposition  that  under  certain  natural  conditions 
unfavorable  to  the  development  of  the  anthrax  bacillus 
an  attenuation  of  its  virulence  takes  place,  and  then, 
again,  an  increase  of  virulence  as  the  condition  becomes 
more  favorable — a  result  which  can  be  produced  by  arti- 
ficial means.  But  whether  this  actually  occurs  we  do 
not  know. 

Prophylaxis  Against  Anthrax  Infection.  Numerous 
investigations  have  been  undertaken  with  the  object  of 
preventing  infection  from  anthrax.  The  efforts  of  Pas- 
teur to  effect  immunity  in  animals  by  preventive  inocu- 
lations of  "  attenuated  virus"  of  the  anthrax  bacillus, 
opened  a  new  field  of  productive  original  research.  Fol- 
lowing in  his  wake  many  others  have  prepared  methods 
of  immunization  against  anthrax  infection;  but  the  one 
adopted  by  Pasteur,  Chamberland  and  Roux  has  alone 
been  practically  employed  on  a  large  scale.  According 
to  these  authors,  two  anthrax  cultures  of  different  de- 
grees of  virulence,  attenuated  by  cultivation  at  42°  to 
43°  C.,  are  used  for  inoculation.  Vaccine  No.  1  kills 
mice,  but  not  guinea-pigs;  Vaccine  No.  2  kills  guinea- 
pigs,  but  not  rabbits,  according  to  Koch,  Gaffky,  and 
Loffler.  The  animals  to  be  inoculated — viz.,  sheep 
and  cattle — are  first  given  a  subcutaneous  injection 
of  one  to  several  tenths  of  a  cubic  centimetre  of 
a  four-day-old  bouillon  culture  of  Vaccine  No.  1; 
after  ten  to  twelve  days  they  receive  a  similar  dose 
of  Vaccine  No.  2.  Prophylactic  inoculations  given 
in  this  way  have  been  widely  employed  in  France, 
Hungary,  and  Russia.  Statistics  collected  by  Cham- 
berland of  the  results  of  twelve  years7  use  of  this 

36 


562  BACTERIOLOGY. 

method  in  France  show  that  3,300,000  sheep  have 
been  thus  inoculated.  Of  these  1  per  cent,  only  have 
died  from  anthrax,  either  during  or  after  treatment; 
whereas  the  mortality  previous  to  the  introduction  of 
this  method  was  10  per  cent,  on  the  average.  Of 
438,000  cattle  inoculations  only  0.33  per  cent,  have 
died;  the  previous  mortality  from  anthrax  was  5  per 
cent.  These  figures  would  seem  to  indicate  the  prac- 
tical value  of  Pasteur's  method  of  inoculation,  notwith- 
standing the  arguments  which  have  been  put  forward  in 
opposition  to  it.  It  is,  however,  not  unattended  with 
danger,  as  some  of  the  animals  succumb  to  the  after- 
effects of  the  attenuated  culture. 

Differential  Diagnosis.  The  differential  diagnosis  of 
the  anthrax  bacillus  is  ordinarily  not  difficult,  as  this 
organism  presents  morphological,  biological,  and  patho- 
gen ical  characteristics  which  distinguish  it  from  all  other 
bacteria.  In  the  later  stages  of  the  disease,  however, 
the  bacilli  may  be  absent  or  difficult  to  find,  and  culti- 
vation on  artificial  media  and  experimental  inoculation 
in  animals  are  not  always  followed  by  positive  results. 
Even  in  sections  taken  from  the  extirpated  pustule  it  is 
sometimes  difficult  to  detect  the  bacilli.  In  such  cases 
only  a  probable  diagnosis  of  anthrax  can  be  made.  It 
should  be  remembered  that  the  bacilli  are  not  found  in 
the  blood  until  shortly  before  death,  and  then  only  in 
varying  quantity;  thus  blood  examinations  often  give 
negative  results,  though  the  bacilli  may  be  present  in 
large  numbers  in  the  spleen,  kidneys,  and  other  organs 
of  the  body.  The  suspected  material  should  be  inocu- 
lated in  nutrient  gelatin  and  agar  in  Petri  plates  and 
in  mice. 

Among  other  bacteria  which  may  possibly  be  mis- 


BACILL  US  ANTHRA CIS  SYMPTOMA T1CL     563 

taken  for  anthrax  bacilli  are  the  bacillus  subtilis  and 
the  bacillus  of  malignant  oedema.  The  former  is  dis- 
tinguished by  its  motility,  by  various  cultural  peculi- 
arities, and  by  being  non-pathogenic.  The  latter  differs 
from  the  anthrax  bacillus  in  form  and  motility,  in  being 
decolorized  by  Gram's  solution,  in  being  a  strict  anaer- 
obe, and  in  various  pathogenic  properties. 

The  diagnosis  of  internal  anthrax  in  man  is  by  no 
means  easy,  unless  the  history  points  definitely  to  infec- 
tion in  the  occupation  of  the  individual.  In  cases  of 
doubt  cultures  should  be  made  and  inoculations  per- 
formed in  animals.  According  to  Cornil  and  Babes, 
some  of  these  cases  may  possibly  be  caused  by  organ- 
isms other  than  the  bacillus  of  anthrax. 

BACILLUS   ANTHRACIS   SYMPTOMATICI. 

(Bacillus  of  Symptomatic  Anthrax.) 

Like  the  bacilli  of  anthrax  and  of  malignant  oedema, 
both  of  which  it  resembles  in  other  respects  also,  the 
bacillus  of  symptomatic  anthrax  is  an  inhabitant  of  the 
soil.  It  is  found  as  the  chief  cause  of  the  disease  in 
animals — principally  cattle  and  sheep — affected  with 
what  is  known  as  "  black  leg,"  "  quarter  evil,"  or 
symptomatic  anthrax  (German,  rauschbrand;  French, 
charbon  symptomatique),  a  disease  which  prevails  in 
certain  localities  in  summer,  and  is  characterized  by  a 
peculiar  emphysematous  swelling  of  the  subcutaneous 
tissues  and  muscles,  especially  over  the  quarters. 

Morphology.  Bacilli  having  rounded  ends,  from 
0.5/j.  to  O.Qfj.  broad  and  from  3//  to  6/J.  long;  mostly 
isolated,  also  occurring  in  pairs,  joined  end-to-end, 
but  never  growing  out  into  long  filaments,  as  the  an- 


564  SA  CTERIOL  OGY. 

thrax  bacilli  in  culture  and  the  bacilli  of  malignant 
oedema  in  the  bodies  of  animals  are  frequently  seen  to 
do.  Under  the  hanging  drop  the  bacilli  are  observed 
to  be  actively  motile,  and  in  stained  preparations  flagella 
may  be  demonstrated  surrounding  the  periphery.  The 
spores  are  elliptical  in  shape,  usually  thicker  than  the 
bacilli,  lying  near  the  middle  of  the  rods,  but  rather 
toward  one  extremity.  This  gives  to  the  bacilli  con- 
taining spores  a  somewhat  spindle  shape. 

Stains  with  the  ordinary  aniline  dyes,  but  not  with 
Gram's  method,  or  only  with  difficulty  and  after  long 
treatment  or  intense  colors. 

Biological  Characters.  Like  the  bacillus  of  malignant 
oedema  this  is  also  a  strict  anaerobe,  and  cannot  be  cul- 
tivated in  an  atmosphere  in  which  oxygen  is  present. 
It  grows  best  under  hydrogen,  and  does  not  grow  under 
carbonic  acid.  This  bacillus  develops  at  the  room-tem- 
perature in  the  usual  culture  media,  in  the  absence  of 
oxygen,  but  it  grows  best  in  those  to  which  1.5  to  2  per 
cent,  of  glucose  or  5  per  cent,  of  glycerin  has  been  added. 

Growth  on  Agar.  The  colonies  on  agar  are  some- 
what more  compact  than  those  of  malignant  oedema,  but 
they  also  send  out  projections  very  often.  In  agar  stick 
cultures,  in  the  incubator,  growth  occurs  after  a  day  or 
two  also  some  distance  below  the  surface,  and  is  accom- 
panied by  the  production  of  gas  and  a  peculiar  disagree- 
able acid  odor. 

Pathogenesis.  The  bacillus  of  symptomatic  anthrax 
is  pathogenic  for  cattle  (which  are  immune  against  ma- 
lignant oedema),  sheep,  goats,  guinea-pigs,  and  mice; 
horses,  asses,  and  white  rats  when  inoculated  with  a  cul- 
ture of  this  bacillus  present  only  a  limited  reaction;  and 
rabbits,  swine,  dogs,  cats,  chickens,  ducks,  and  pigeons 


BACILLUS  ANTHEACIS  SYMPTOMATIC!.     565 

are,  as  a  rule,  naturally  immune  to  the  disease.  The 
guinea-pig  is  the  most  susceptible  of  test  animals. 
When  susceptible  animals  are  inoculated  subcutane- 
ously  with  pure  cultures  of  this  organism,  with  spores 
attached  to  a  silk  thread,  or  with  bits  of  tissue  from 
the  affected  parts  of  another  animal  dead  of  the  disease, 
death  ensues  in  from  twenty-four  to  thirty-six  hours. 
At  the  autopsy  a  bloody  serum  is  found  in  the  subcuta- 
neous tissues  extending  from  the  point  of  inoculation 
over  the  entire  surface  of  the  abdomen,  and  the  muscles 
present  a  dark-red  or  black  appearance,  even  more  in- 
tense in  color  than  in  malignant  oedema,  and  there  is  a 
considerable  development  of  gas.  The  lymphatic  glands 
are  markedly  hypenemic. 

The  disease  occurs  chiefly  in  cattle,  more  rarely  in 
sheep  and  goats;  horses  are  not  attacked  spontaneously 
— i.  e.y  by  accidental  infection.  In  man,  infection  has 
never  been  produced,  though  ample  opportunity,  by 
infection  through  wounds  in  slaughter-houses  and  by 
the  ingestion  of  infected  meat,  has  been  given.  The 
usual  mode  of  natural  infection  by  symptomatic  an- 
thrax is  through  wounds  which  penetrate  not  only  the 
skin  but  the  deep  intercellular  tissues;  some  cases  of 
infection  by  ingestion  have  been  observed.  The  patho- 
logical findings  present  the  conditions  above  described 
as  occurring  in  experimental  infection. 

Symptomatic  anthrax,  like  anthrax  and  malignant 
oedema,  is  a  disease  of  the  soil,  but  it  shows  a  more 
limited  endemic  distribution  than  the  first,  and  is  differ- 
ently distributed  over  the  earth's  surface  than  the  sec- 
ond of  these  diseases,  being  confined  especially  to  places 
over  which  infected  herds  of  cattle  have  been  pastured. 
It  is  doubtful  whether  the  bacilli  are  capable  of  devel- 


566  BACTERIOLOGY. 

opment  outside  of  the  body  like  anthrax.  In  the  form 
of  spores,  however,  reproduction  may  take  place;  and 
by  contamination  with  these,  through  deep  wounds  ac- 
quired by  animals  in  infected  pastures,  the  disease  is 
spread.  Possibly  also  it  may  originate  through  infec- 
tion of  the  mouth  and  by  feeding — which  would  account 
for  the  cases  of  symptomatic  anthrax  occurring  in  stall- 
fed  cattle  (Hafner). 

It  is  well  known  to  veterinarians  that  recovery  from 
one  attack  of  symptomatic  anthrax  protects  an  animal 
against  a  second  infection.  Artificial  immunity  to  in- 
fection can  also  be  produced  in  various  ways  :  by  intra- 
venous inoculations;  or,  in  guinea-pigs,  by  inoculations 
with  bouillon  cultures  which  have  been  kept  for  a  few 
days  and  as  a  result  have  lost  their  original  virulence, 
or  with  cultures  kept  in  an  incubating  oven  at  a  tem- 
perature of  42°  to  43°  C.;  or  by  inoculations  made  into 
the  extremity  of  the  tail;  or  by  inoculations  with  filtered 
cultures,  or  with  cultures  sterilized  by  heat.  For  the 
production  of  immunity  in  cattle,  Arloing,  Cornevin, 
and  Thomas  recommend  the  use  of  a  dried  powder  of 
the  muscles  of  animals  which  have  succumbed  to  the 
disease,  and  which  have  been  subjected  to  a  suitable 
temperature  to  ensure  attenuation  of  the  virulence  of 
the  spores  contained  in  it.  Two  vaccines  are  prepared, 
as  in  anthrax — a  stronger  vaccine  by  exposure  of  a  por- 
tion of  the  powder  to  a  temperature  of  85°  to  90°  C. 
for  six  hours,  and  a  weaker  vaccine  by  exposure  for  the 
same  time  to  a  temperature  of  100°  to  104°  C.  Inocu- 
lations made  with  this  attenuated  virus  (into  the  end  of 
the  tail) — the  weakest  first  and  later  the  stronger — give 
rise  to  a  local  reaction  of  moderate  intensity,  and  the 
animal  is  subsequently  immune  from  the  effects  of  the 


BA  CILL  US  ANTHRA  CIS  S  YMPTOMA  TICI.     567 

most  virulent  material  and  from  the  disease.    Fourteen 

• 

days  are  allowed  to  elapse  between  the  two  inoculations. 
The  results  obtained  from  this  method  of  preventive  in- 
oculation seem  to  have  been  very  satisfactory.  Accord- 
ing to  the  statistics  of  Hafner,  Luchanka,  Hess,  Strebel, 
etc.  (1885-93),  including  many  thousand  cattle  treated, 
the  mortality,  which  among  22,300  non-inoculated  cattle 
was  2.20  per  cent.,  has  been  reduced  to  0.16  per  cent, 
in  14,700  animals  inoculated. 

To  recapitulate  briefly,  the  principal  points  of  differ- 
entiation between  this  bacillus  and  the  bacillus  of  malig- 
nant oedema,  which  it  closely  simulates,  are  smaller;  it 
does  not  develop  into  long  threads  in  the  tissues;  it  is 
more  actively  motile,  and  forms  spores  more  readily  in 
the  animal  body  than  does  the  bacillus  of  malignant 
oedema.  It  is  pathogenic  for  cattle,  while  malignant 
oedema  is  not;  and  swine,  dogs,  rabbits,  chickens,  and 
pigeons,  which  are  readily  infected  with  malignant 
oedema,  are  not,  as  a  rule,  susceptible  to  symptomatic 
anthrax. 


CHAPTER   XXXIII. 

SPIRILLUM   CHOLERA   ASIATICS   (KOCH?S   COMMA 
BACILLUS   OF   ASIATIC   CHOLERA). 

IN  1883,  Koch  separated  a  characteristically  curved 
organism  from  the  dejecta  and  intestines  of  cholera 
patients — the  so-called  "  comma  bacillus."  This  he 
declared  to  be  absent  from  the  stools  and  intestinal  con- 
tents of  healthy  persons  and  of  persons  suffering  from 
other  affections.  The  organism  was  said  to  possess  cer- 
tain morphological  and  biological  features  which  readily 
distinguished  it  from  all  previously  described  organisms. 
It  was  absent  from  the  blood  and  viscera,  and  was  found 
only  in  the  intestines;  and  in  greater  number,  it  was 
said,  the  more  acute  the  attack.  Koch  also  demonstrated 
an  invasion  of  the  mucosa  and  its  glands  by  the  comma 
bacilli.  The  organisms  were  found  in  the  stools  on 
staining  the  mucous  flakes  or  the  fluid  with  methylene- 
blue  or  fuchsin,  and  sometimes  alone;  by  means  of  cul- 
tivation on  gelatin  they  were  readily  separated  from  the 
stools.  During  his  stay  in  India,  in  Egypt,  and  at  Tou- 
lon, Koch  had  examined  over  one  hundred  cases,  and 
other  investigators  confirmed  his  statements.  Numer- 
ous control  observations  made  upon  other  diarrhoeic  de- 
jecta and  upon  normal  stools  were  negative;  the  comma 
bacillus  was  found  in  choleraic  material  only,  or  in  mate- 
rial contaminated  by  cholera.  Soon  other  observers, 
however,  described  comma-shaped  organisms  of  non- 


SPIRILLUM  CHOLERA  ASIATICS.          569 

choleraic  origin;  Finkler  and  Prior,  for  instance,  found 
them  in  the  diarrhceic  stools  of  cholera  nostras,  Deneke 
in  cheese,  Lewis  and  Miller  in  saliva.  All  of  these 
organisms,  however,  differed  in  many  respects  from 
Koch's  comma  bacillus,  and  since  then  it  has  been 
proved  that  none  of  them  was  affected  by  the  specific 
serum  of  animals  immunized  to  cholera;  and  gradually 
the  exclusive  association  of  Koch's  vibrio  with  cholera 

FIG.  74.  FIG.  75. 


Contact  smear  of  colony  of  Contact  spirilla  preparation  from  plate 

cholera   spirilla   from  agar.  culture   of   cholera.      X    800   diameters. 

X  700  diameters.  (DUNHAM.)          (DUNHAM.) 

became  almost  generally  acknowledged.  It  is  now  re- 
garded by  bacteriologists  everywhere  to  be  the  specific 
cause  of  Asiatic  cholera. 

Morphology.  Curved  rods  with  rounded  ends  which 
do  not  lie  in  the  same  plane,  from  0.8//  to  2/*  in  length 
and  about  0. 4//  in  breadth.  The  curvature  of  the  rods 
may  be  very  slight,  like  that  of  a  comma,  or  distinctly 
marked,  particularly  in  fresh  unstained  preparations 
of  full-grown  individuals,  presenting  the  appearance 
of  a  half -circle.  By  the  junction  of  two  vibrios 
S-shaped  forms  are  produced,  and  under  unfavorable 


570  BACTERIOLOGY. 

conditions  of  growth  they  may  develop  into  long,  spiral 
filaments,  which  may  consist  of  numerous  spiral  turns 
in  which  it  is  impossible  to  recognize  any  connection 
with  the  individual  elements  of  which  they  are  made 
up.  In  stained  preparations  the  spiral  character  of  the 
long  filaments  is  often  obliterated,  or  nearly  so.  Under 
favorable  conditions  of  growth — that  is,  when  the 
growth  is  rapid  —the  short-curved  or  almost  straight 
forms  are  commonly  observed  (Figs.  74  and  75).  In 
old  cultures  involution  forms  are  frequent. 

Stains  with  the  aniline  colors  usually  employed,  but 
not  as  readily  as  many  other  bacteria;  an  aqueous  solu- 
tion of  carbol-fuchsin  is  recommended  as  the  most  reli- 
able staining  agent  with  the  application  of  a  few  minutes' 
heat.  It  is  decolorized  by  Gram's  method.  The  motile 
organs  exhibit  one  or  two  long,  fine,  spiral  flagellse 
attached  to  one  end  of  the  rods. 

Biological  Characters.  An  aerobic  (facultative  ana- 
erobic), liquefying,  motile  spirillum.  Grows  readily  in 
the  ordinary  culture  media,  best  at  37°  C.,  but  also  at 
the  room-temperature  (22°  C.);  does  not  grow  at  a  tem- 
perature above  42°  or  below  8°  C.  Does  not  form 
spores. 

In  gelatin  plate  cultures,  at  22°  C.,  at  the  end  of 
twenty-four  hours,  small,  round,  yellowish-white  to  yel- 
low colonies  may  be  seen  in  the  depths  of  the  gelatin, 
which  later  grow  toward  the  surface  and  cause  liquefac- 
tion of  the  medium,  the  colonies  lying  at  the  bottom  of 
the  holes  or  pocket  thus  formed.  The  zone  of  lique- 
faction, which  increases  rapidly,  remains  at  first  clear, 
then  becomes  cloudy,  mostly  gray,  as  the  result  of  the 
growth  of  the  colonies.  In  many  cases  after  a  time 
concentric  rings,  which  increase  from  day  to  day,  appear 


SPIRILLUM  CHOLERA  ASIATICS.  571 

in  the  zone  of  liquefaction.  (See  Figs  76  and  77.) 
Examined  under  a  low-power  lens,  at  the  end  of  sixteen 
to  twenty-four  hours,  the  colonies  appear  as  small,  light 
yellow,  round,  coarsely  granular  disks,  with  a  more  or 
less  irregular  outline.  In  many  cases  at  this  stage  an 

FIG.  76.  FIG.  77. 


Cholera  colonies  in  gelatin ; 
Cholera    colonies    in    gelatin;   twenty-four    thirty-six  to  forty-eight  hours' 
hours' growth.    (DUNHAM.)  growth.  X  about  30  diameters. 


ill-defined  halo  is  seen  to  surround  the  granular  colony, 
which  by  transmitted  light  has  a  peculiar  reddish  tint. 
The  older  the  colonies  become  the  more  the  granular 
structure  increases,  until  a  stage  is  reached  when  the 
surface  looks  as  if  it  were  covered  with  little  fragments 
of  broken  glass  (Koch).  Liquefaction  continues  around 
the  colonies,  their  structure  appears  fissured  and  coarsely 
granular  in  texture,  and  occasionally  a  hair-like  border 
is  formed  at  the  periphery  (Fig.  78),  or  a  gray  trans- 
parent zone,  until  the  entire  colony  breaks  up  into  frag- 
ments. Sometimes  the  colonies  may  be  retained  as  com- 
pact masses  in  the  zone  of  liquefaction,  and  then  they  are 


572  BACTERIOLOGY. 

dark-yellow  or  brown  in  color,  and  forms  occur  which 
are  absolutely  unlike  the  typical  cholera  colonies.  In 
gelatin  stick  cultures  the  growth  is  at  first  thread-like 
and  uncharacteristic.  At  the  end  of  twenty-four  to 
thirty-six  hours  a  small,  funnel-shaped  depression  ap- 
pears on  the  surface  of  the  gelatin,  which  soon  spreads 
out  in  the  form  of  an  air-bubble  above,  while  below 
this  is  a  whitish,  viscid  mass.  Later,  the  funnel  in- 
creases in  depth  and  diameter,  and  at  the  end  of  from 
four  to  six  days  may  reach  the  edge  of  the  test-tube; 

PIG.  78. 


Cholera  colony  in  gelatin.    X  30  diameters.    (DUNHAM.) 

in  from  eight  to  fourteen  days  the  upper  two-thirds  of 
the  gelatin  is  completely  liquefied.  (See  Fig.  79  and 
Fig.  31,  page  230.)  Freshly  isolated  cholera  vibrios 
liquefy  gelatin  more  rapidly  than  old  laboratory  cul- 
tures; a  certain  variation  in  the  characteristic  liquefac- 
tion of  the  gelatin  even  in  fresh  cultures  under  some 
circumstances  should  be  borne  in  mind  in  making  a 
diagnosis.  Such  variations  in  cultural  peculiarities 
occur  also  with  other  bacteria,  and  only  the  sum  of  all 
the  characteristics  taken  together  enables  a  positive 
diagnosis  to  be  established. 

Upon  the  surface  ofagar  the  comma  bacillus  develops 
a  moist,  shining,  grayish-yellow  layer.  Blood-serum 
is  rapidly  liquefied  at  the  temperature  of  the  incu- 


SPIRILLUM  CHOLERA  ASIATICS. 


573 


bator.  In  bouillon  the  growth  is  rapid  and  abun- 
dant; in  the  incubator  at  the  end  of  ten  to  sixteen  hours 
the  liquid  is  diffusely  clouded,  and  on  the  surface  a 
wrinkled  membranous  layer  is  often  formed.  In  gen- 


FIG.  79. 


A  characteristic  series  of  cholera  cultures  in  gelatin ;  one,  two,  three,  four, 
and  six  days'  growth.    (DUNHAM.) 

eral  the  spirillum  grows  in  any  liquid  containing  a 
small  quantity  of  organic  matter  and  having  a  slightly 
alkaline  reaction.  An  acid  reaction  of  the  culture  me- 
dium prevents  its  development,  as  a  rule;  but  it  has  the 
power  of  gradually  accommodating  itself  to  the  pres- 
ence of  vegetable  acids,  and  grows  upon  potatoes,  in 
the  incubator  only,  which  have  a  slight  acid  reaction. 
Abundant  development  occurs  in  bouillon  which  has 
been  diluted  with  eight  to  ten  parts  of  water  and  in 


574  BACTERIOLOGY. 

simple  peptone  solution,  and  it  has  been  shown  by  ex- 
periment that  it  also  multiplies  to  some  extent  in  steril- 
ized river  or  well-water,  and  preserves  its  vitality  in 
such  water  for  several  weeks  or  even  months.  Koch 
found  in  his  early  investigations  that  rapid  multiplica- 
tion may  occur  upon  the  surface  of  moist  linen,  and  also 
demonstrated  the  presence  of  this  spirillum  in  the  foul 
water  of  a  tank  in  India  which  was  used  by  the  natives 
for  drinking  purposes. 

The  comma  bacillus  belongs  to  the  class  of  aerobic 
organisms,  inasmuch  as  it  grows  readily  only  in  the  pres- 
ence of  oxygen,  and  that  it  develops  active  motility 
only  when  a  certain  amount  of  oxygen  is  present.  It 
does  not  grow  in  the  total  absence  of  oxygen,  but  a 
small  quantity  of  oxygen  is  all  that  is  required  for  its 
development,  as  in  the  intestines. 

Temperature  is  also  of  considerable  importance  in  the 
growth  of  cultures.  Active  growth  does  not  begin 
until  a  temperature  of  22°  to  25°  C.  is  reached,  though 
the  optimum  growth  is  between  30°  and  40°  C. 

The  vitality  of  cultures  of  the  comma  bacillus  is 
quickly  destroyed  by  desiccation.  If  a  culture  be 
spread  on  a  cover-glass  and  exposed  to  the  action  of 
the  air  at  room-temperature  the  bacilli  are  dead  at  the 
end  of  two  or  three  hours,  unless  the  layer  of  culture 
is  very  thick,  when  it  may  take  twenty-four  hours  or 
more  to  kill  all  the  bacilli.  This  fact  indicates  that 
infection  is  not  produced  by  means  of  dust  or  other  dried 
objects  contaminated  with  cholera  bacilli.  The  trans- 
misson  of  these  organisms  through  the  air,  therefore, 
can  only  take  place  for  short  distances,  as  by  a  spray  of 
infectious  liquids  by  mechanical  means — as,  for  instance, 


SPIRILLUM  CHOLERA  ASIATICS.  575 

the  breaking  of  waves  in  a  harbor,  on  water-wheels, 
etc.,  or  in  moist  wash  of  cholera  patients. 

The  cholera  bacillus  is  also  injuriously  affected  by  the 
abundant  growth  of  saprophytic  bacteria.  It  is  true 
that  when  associated  with  other  bacteria,  if  present  in 
large  numbers,  and  if  the  conditions  for  their  develop- 
ment are  particularly  favorable,  the  cholera  bacillus 
may  at  first  gain  the  upper  hand,  as  in  the  moist  linen 
of  cholera  patients,  or  in  soil  impregnated  with  chol- 
era dejecta;  but  later,  after  two  or  three  days,  even  in 
such  cases,  the  bacilli  die  off  and  other  bacteria  gradu- 
ally take  their  place.  Thus  Koch  found  that  the  fluid 
contents  of  privies  twenty-four  hours  after  the  introduc- 
tion of  comma  bacilli  no  longer  contained  the  living 
organisms;  in  Berlin  canal-water  they  were  not  demon- 
strable for  more  than  six  to  seven  days,  as  a  rule.  In 
the  dejecta  of  cholera  patients  they  were  found  usually 
only  for  a  few  days  (one  to  three  days),  though  rarely 
they  have  been  observed  for  twenty  to  thirty  days,  and 
on  one  occasion  for  one  hundred  and  twenty  days.  In 
unsterilized  water  they  may  also  retain  their  vitality  for 
a  relatively  long  time  ;  thus  in  stagnant  well-water  they 
have  been  found  for  eighteen  days,  and  in  an  aquarium 
containing  plants  and  fishes,  the  water  of  which  was 
inoculated  with  cholera  germs,  they  were  isolated  sev- 
eral months  later  from  the  mud  at  the  bottom.  In 
running  river-water,  however,  they  have  not  been  ob- 
served for  over  six  to  eight  days.  Even  in  cultures  the 
comma  bacillus  is  one  of  the  shorter-lived  bacteria. 
They  have  been  observed,  however,  in  pure  bouillon 
cultures  for  three  to  four  months,  and  in  agar  cul- 
tures for  six  months,  and  occasionally  in  one-year- 
old  cultures  when  they  were  protected  from  desicca- 


576  BACTERIOLOGY. 

tion.  In  these  they  occurred  only  in  involution 
forms. 

The  comma  bacillus  is  killed  by  exposure  to  moist 
heat  at  60°  C.  in  ten  minutes.  The  bacilli  have  been 
found  alive  in  ice  kept  for  a  few  days,  but  ice  which 
has  been  preserved  for  several  weeks  does  not  contain 
living  bacilli. 

Chemical  disinfectants  readily  destroy  the  vital- 
ity of  cholera  vibrios.  For  disinfection  on  a  small 
scale,  as  for  washing  the  hands  when  contaminated  with 
cholera  infection,  a  0. 1  per  cent,  solution  of  bichloride 
of  mercury  or  a  2  to  3  per  cent,  solution  of  carbolic  acid 
may  be  used.  For  disinfection  on  a  large  scale,  as  for 
the  disinfection  of  cholera  stools,  strongly  alkaline  milk 
of  lime,  according  to  PfuhFs  experiments,  is  an  excel- 
lent agent.  The  wash  of  cholera  patients,  contaminated 
furniture,  floors,  etc.,  may  be  disinfected  by  a  solution 
of  5  per  cent,  carbolic  acid  and  soap-water.  The  dis- 
infecting action  of  mineral  acids,  particularly  of  sul- 
phuric acid,  has  been  advantageously  employed  for  the 
disinfection  of  entire  systems  of  water-works  into  which 
cholera  bacilli  had  gained  access. 

Pohl,  Bujivid,  and  Dunham  have  shown  that  when 
a  small  quantity  of  chemically  pure  sulphuric  acid  is 
added  to  a  twenty-four-hour  bouillon  culture  of  the 
cholera  bacillus  containing  peptone  a  reddish-violet 
color  is  produced.  Brieger  separated  the  pigment 
formed  in  this  reaction — the  so-called  cholera-red — and 
showed  that  it  was  indol,  and  that  the  reaction  was 
nothing  more  than  the  well-known  indol  reaction.  Sal- 
kowski  and  Petri  then  demonstrated  that  the  cholera 
bacilli  produced  in  thin  bouillon  cultures,  along  with 
indol,  nitrites  by  reduction  from  the  nitrates  con- 


SPIRILLUM  CHOLERA  ASIATICS.  577 

tained  in  small  quantities  in  the  culture  media;  and 
showed  that  it  is  the  setting  free  of  nitric  acid,  upon 
the  addition  of  sulphuric  acid  to  the  culture,  which 
gives  with  indol  the  red  body  upon  which  the  cholera 
reaction  depends.  For  a  long  time  it  was  believed  that 
this  nitroso-indol  reaction  was  peculiar  to  the  cholera 
bacillus,  and  great  weight  was  placed  on  it  as  a  diag- 
nostic test.  It  has  since  been  shown,  however,  that 
there  are  a  number  of  other  vibrios  which,  under  sim- 
ilar conditioDS  as  the  cholera  vibrio,  give  the  same  red 
reaction.  The  reaction  is,  nevertheless,  a  constant  and 
characteristic  peculiarity  of  this  spirillum,  and  is  of 
unquestionable  value.  It  is  even  more  valuable  as  a 
negative  than  as  a  positive  test,  as  the  absence  of  the 
reaction  enables  one  to  say  of  a  suspected  organism  that 
it  is  not  the  cholera  spirillum.  There  are,  however, 
certain  precautions  to  be  observed  in  its  use.  It  has 
been  shown  that  the  reaction  may  be  absent,  for  in- 
stance, when  the  culture  contains  either  too  much  or 
too  little  nitrate.  It  is,  therefore,  advisable  not  to  em- 
ploy a  bouillon  culture  the  composition  of  which  is 
uncertain,  but  a  distinctly  alkaline  solution  of  peptone, 
containing  1  per  cent,  pure  peptone  and  0.5  per  cent, 
of  pure  chloride  of  sodium  (Dunham's  solution).  With 
such  a  solution  constant  results  can  be  obtained. 

Pathogenesis.  Since  none  of  the  lower  animals  is  nat- 
urally subject  to  cholera,  nor  has  ever  contracted  the 
disease  during  the  prevalence  of  an  epidemic  or  as  the 
result  of  the  iugestion  of  food  contaminated  with  chol- 
eraic excreta,  there  is  no  reason  to  expect  that  inocula- 
tions of  pure  cultures  of  the  spirillum,  either  subcuta- 
neously  or  by  the  mouth,  will  give  rise  in  animals  to  a 
typical  attack  of  cholera.  It  has  been  shown,  more- 

37 


578  BACTERIOLOGY. 

over,  that  the  comma  bacillus  is  extremely  sensitive  to 
the  action  of  acids,  and  is  quickly  destroyed  by  the  acid 
secretions  of  the  stomach  of  man  or  the  lower  animals 
when  these  secretions  are  normally  produced.  Despite 
the  small  prospects  of  success,  however,  from  animal 
experiments,  these  have  been  undertaken  again  and 
again,  until  finally  a  method  was  found  by  which  at 
least  similar  processes  have  been  produced  in  test  ani- 
mals by  inoculation  of  pure  cultures  of  the  cholera 
vibrio.  Koch  sought  to  produce  infection  in  guinea- 
pigs  per  vias  naturales  by  first  neutralizing  the  con- 
tents of  the  stomach  with  a  solution  of  carbonate  of 
soda — 5  c.c.  of  a  5  per  cent,  solution  injected  into  the 
stomach  through  a  pharyngeal  catheter — and  then  after 
a  while  administered  through  a  similar  catheter  10  c.c. 
of  a  liquid  into  which  had  been  put  one  or  two  drops 
of  a  bouillon  culture  of  the  comma  bacillus.  The  ani- 
mal then  receives  a  dose  of  1  c.c.  of  tincture  of  opium 
per  200  grammes  of  body-weight  introduced  into  the 
abdominal  cavity,  for  the  purpose  of  controlling  the 
peristaltic  movements.  As  a  result  of  this  treatment 
the  animals  are  completely  narcotized  for  about  half  an 
hour,  but  recover  from  it  without  showing  any  ill  effects. 
On  the  evening  of  the  same  or  following  day  the  ani- 
mal shows  an  indisposition  to  eat  and  other  signs  of 
weakness,  its  posterior  extremities  become  weak  and 
apparently  paralyzed,  and,  as  a  rule,  death  occurs 
within  forty-eight  hours  with  the  symptoms  of  collapse 
and  fall  of  temperature.  At  the  autopsy  the  small 
intestine  is  found  to  be  congested  and  filled  with  a 
watery  fluid  containing  the  spirillum  in  great  numbers. 
Koch  experimented  in  this  way  on  about  one  hundred 
guinea-pigs.  These  results,  however,  are  somewhat 


SPIRILLUM  CHOLERA  ASIATICS.  579 

weakened  by  the  fact  that  experiments  made  with 
some  other  bacteria — viz.,  those  isolated  by  Finkler  and 
Prior,  Deneke,  and  Miller,  and  morphologically  similar 
to  the  comma  bacillus  of  Koch — occasionally  pro- 
duced death  when  introduced  in  the  same  way  into 
the  small  intestines  of  guinea-pigs;  but  while  only 
twelve  out  of  fifty-one  animals  died  when  injected 
with  cultures  of  these  last-mentioned  bacteria,  in  the 
cholera  experiments  there  was  90  per  cent,  of  deaths, 
and  when  larger  doses  were  administered  all  of  the 
animals  died.  Control  experiments  made  with  many 
other  bacteria  gave  negative  results.  Intraperitoneal 
injections  of  larger  quantities  of  pure  cholera  cultures 
also  often  produce  death  in  rabbits  and  mice. 

There  are  several  cases  on  record  which  furnish  the 
most  satisfactory  evidence  that  the  cholera  bacillus  is 
able  to  produce  the  disease  in  man.  In  1884,  a  student 
in  Koch's  laboratory  in  Berlin,  who  was  taking  a  course 
on  cholera,  became  ill  with  a  severe  attack  of  cholera. 
At  that  time  there  was  no  cholera  in  Germany,  and  the 
infection  could  not  have  been  produced  in  any  other 
way  than  through  the  cholera  cultures  which  were  being 
used  for  the  instruction  of  students.  In  1892,  Petten- 
kofer  and  Emmerich  experimented  on  themselves  by 
swallowing  small  quantities  of  fresh  cholera  cultures 
obtained  from  Hamburg.  Pettenkofer  was  affected 
with  a  mild  attack  of  cholerine  or  severe  diarrhoea,  from 
which  he  recovered  in  a  few  days  without  any  serious 
effects;  but  Emmerich  became  very  ill.  On  the  night 
following  the  infection  he  was  attacked  by  frequent  evac- 
uations of  the  characteristic  rice-water  type,  cramps, 
tympanitis,  and  great  prostration.  His  voice  became 
hoarse,  and  the  secretion  of  urine  was  somewhat  dimiu- 


580  BACTERIOLOGY. 

ished,  this  condition  lasting  for  several  days.  In  both 
cases  the  cholera  spirillum  was  obtained  in  pure  cul- 
ture from  the  dejecta.  Another  instance  is  reported 
by  Metschnikoff,  in  Paris,  of  a  man  who  became 
infected  experimentally.  In  this  case  the  algid  stage 
of  cholera  was  produced,  with  complete  suppression  of 
urine,  cramps  in  the  legs,  contraction  of  the  extrem- 
ities, and  collapse,  the  man's  life  being  saved  only  with 
difficulty.  Finally,  there  is  the  case  of  Dr.  Oergel, 
of  Hamburg,  who  accidentally,  while  experimenting  on 
a  guinea-pig,  had  some  of  the  infected  peritoneal  fluid 
to  squirt  into  his  mouth.  He  was  taken  ill  and  died 
a  few  days  afterward  of  typical  cholera,  though  at  the 
time  of  his  death  there  was  no  cholera  in  the  city. 
These  accidents  and  experiments  would  certainly  seem 
to  prove  conclusively  the  capability  of  pure  cholera  cul- 
tures of  producing  the  disease;  and  yet  Strickler  and 
Hasterlick  (Vienna,  1893)  report  negative  results  from 
experiments  on  the  human  subject.  This  only  goes  to 
show,  however,  that  in  cholera,  like  other  infectious 
diseases,  there  is  an  individual  susceptibility.  It  is 
also  possible  that  the  cultures  used  for  experimentation 
may  have  lost  in  virulence,  as  cholera  cultures  are  so 
liable  to  do  when  kept  for  any  length  of  time. 

Cholera  Toxin.  Koch  was  the  first  to  assume,  as  the 
result  of  his  investigations,  that  the  severe  symptoms 
of  the  algid  stage  of  cholera  were  due  to  the  effects  of 
a  toxin  produced  by  the  growth  of  the  comma  bacillus 
in  the  intestines. 

In  1892,  Pfeiffer  published  an  account  of  his  elabo- 
rate researches  relating  to  the  cholera  poison.  He  finds 
that  recent  aerobic  cultures  of  the  cholera  spirillum  con- 
tain a  specific  toxic  substance  which  is  fatal  to  guinea- 


SPIRILLUM  CHOLER^E  ASIATICS.  581 

pigs  in  extremely  small  doses.  This  substance  stands  in 
close  relation  with  the  bacterial  cells,  and  is  perhaps  an 
integral  part  of  them.  The  spirilla  may  be  killed  by 
chloroform,  thymol,  or  by  desiccation  without  apparent 
injury  to  the  toxic  power  of  this  substance.  It  is  de- 
stroyed, however,  by  absolute  alcohol,  by  concentrated 
solutions  of  neutral  salts,  and  by  the  boiling  tempera- 
ture. Secondary  toxic  products  are  formed  which  have 
a  similar  physiological  action,  but  are  from  ten  to  twenty 
times  less  potent.  Similar  toxic  substances  were  ob- 
tained by  Pfeiffer  from  cultures  of  Finkler  and  Prior's 
spirillum  and  from  the  spirillum  Metschnikovi. 

Cholera  Immunity.  Koch  found  in  his  animal  experi- 
ments that  recovery  from  an  intraperitoneal  infection 
with  small  doses  of  living  cholera  vibrios  produced  a 
certain  immunity  against  larger  doses,  though  the  ani- 
mals inoculated  were  not  very  much  more  resistant  to 
the  cholera  poison  than  they  were  originally.  In  1892 
Lazarus  observed  that  the  blood-serum  of  persons  who 
had  recently  recovered  from  an  attack  of  cholera  pos- 
sessed the  power  of  preventing  the  development  in 
guinea-pigs  of  cholera  bacilli,  which  in  these  animals 
are  rapidly  fatal  when  injected  intraperitoneally ;  while 
the  serum  of  healthy  individuals  had  no  such  effect. 
He  attributed  this  to  the  presence  in  the  serum  of  con- 
valescents from  cholera  of  antitoxic  substances  which 
neutralized  the  poison  produced  by  the  cholera  vibrios, 
in  the  same  manner  as  the  antitoxins  of  diphtheria  and 
tetanus.  Pfeiffer,  on  the  other  hand,  maintained  that 
the  serum  contained  bactericidal  substances  which  killed 
the  bacilli  so  rapidly  when  injected  into  the  animal  that 
they  did  not  have  time  to  produce  their  specific  poison, 
and  that  thus  the  death  of  the  animal  was  prevented. 


582  BACTERIOLOGY. 

The  serum  is  now  known  to  be  feebly  antitoxic  and 
strongly  bactericidal.  This  specific  change  in  the  blood 
is  observed  to  take  place  from  eight  to  ten  days  after 
the  termination  of  an  attack  of  cholera  and  reaches  its 
maximum  during  the  fourth  week  of  convalescence, 
after  which  it  declines  rapidly  and  disappears  entirely 
in  about  two  or  three  months.  Similar  antitoxic  or 
bactericidal  substances  exist  also  in  the  serum  of  guinea- 
pigs,  rabbits,  and  goats,  when  these  animals  are  immu- 
nized artificially  against  cholera  by  subcutaneous  or 
intraperitoneal  injections  of  living  or  dead  cultures. 
These  specific  substances  present  in  the  blood  of  chol- 
era-immune men  and  animals  act  only  upon  organisms 
similar  to  those  with  which  they  were  infected  ;  but, 
as  Pfeiffer  showed,  this  specific  relation,  which  is  found 
to  exist  between  the  antibacterial  and  protective  sub- 
stances produced  during  immunization  and  the  bacteria 
employed  to  immunize  the  animals,  is  not  confined 
alone  to  cholera.  The  discovery,  moreover,  of  this 
specific  reaction  of  the  blood-serum  of  immunized  men 
and  animals  when  brought  in  contact  with  the  spirilla, 
has  given  us  an  apparently  reliable  means  of  distin- 
guishing the  cholera  from  all  other  vibrios,  and  the 
disease  cholera  from  other  similar  affections,  both  of 
which  have  proved  to  be  of  great  value,  particularly  in 
obscure  or  doubtful  cases,  in  which  heretofore  the  only 
method  of  differential  diagnosis  available — viz.,  by 
cultural  tests — was  often  unsatisfactory. 

Cholera  in  man  is  an  infective  process  of  the  epithe- 
lium of  the  intestine,  in  which  the  spirilla  clinging  to 
and  between  the  epithelial  cells  produce  a  partial  or 
entire  necrosis  and  final  destruction  of  the  epithelial 
covering,  which  thus  renders  possible  the  absorption  of 


SPIRILLUM  CHOLERA  ASIATICS.  583 

the  cholera  toxin  formed  by  the  growth  of  the  spirilla. 
The  larger  the  surface  of  the  mucous  membrane  infected, 
the  more  luxuriant  the  development  of  bacilli  and  the 
production  of  toxin,  the  more  pronounced  will  be  the 
poisoning,  ending  fatally  in  a  toxic  paralysis  of  the 
circulatory  and  thermic  centres.  On  the  other  hand, 
however,  there  may  be  cases  where,  in  spite  of  the  large 
number  of  cholera  bacilli  present  in  the  dejecta,  severe 
symptoms  of  intoxication  may  be  absent.  In  such  cases 
the  destruction  of  epithelium  is  then  either  not  pro- 
duced or  so  slight  that  the  toxic  substance  absorbed  is 
not  in  sufficient  concentration  to  give  rise  to  the  algid 
stage  of  the  disease,  or  for  some  reason  the  toxin  is 
not  produced  to  any  extent  by  the  spirilla.  In  no 
stage  of  the  disease  are  living  cholera  spirilla  found  in 
the  organs  of  the  body  or  in  the  secretions. 

From  this  fact  and  other  known  properties  of  the 
cholera  bacillus,  which  have  already  been  referred  to, 
several  important  deductions  may  be  made  with  regard 
to  the  mode  of  transmission  of  cholera  infection.  In 
the  first  place  the  bacilli  evidently  leave  the  bodies  of 
cholera  patients,  chiefly  in  the  dejections  during  the  early 
part  of  the  disease  (they  have  usually  disappeared  after 
the  fourth  to  the  fourteenth  day),  and  only  these  dejec- 
tions, therefore,  and  objects  contaminated  by  them,  such 
as  bed  and  body  wash,  floors,  vaults,  soil,  well-water 
and  river-water,  etc.,  can  be  regarded  as  possible  sources 
of  infection.  There  is  a  special  limitation  even  in 
these  sources  of  infection,  owing  to  the  fact  that  this 
spirillum  is  so  easily  destroyed  by  desiccation  and 
crowded  out  by  saprophytic  organisms.  Thus,  as  a 
rule,  only  fresh  dejections  and  freshly  contaminated 
objects  are  liable  to  convey  infection;  after  they  have 


584  BACTERIOLOGY. 

become  completely  dry  there  is  little  danger.  Farther, 
we  must  conclude  from  the  distribution  of  the  cholera 
bacillus  in  the  body  and  from  experiments  upon  ani- 
mals that  the  commonest  mode  of  infection  is  by  way 
of  the  mouth,  and  chiefly  by  means  of  water  used  for 
drinking  purposes,  for  the  preparation  of  food,  etc.  In 
recent  times  cholera  spirilla  have  been  found  not  infre- 
quently in  water  (wells,  water-mains,  rivers,  harbors, 
and  canals)  which  have  become  contaminated  by  the 
dejections  of  cholera  patients. 

But,  like  other  infectious  diseases,  not  everyone  who 
is  exposed  to  infection  is  attacked  by  cholera.  The 
bacilli  have  been  found  during  cholera  epidemics  in  the 
dejections  of  healthy  individuals  without  any  patholog- 
ical symptoms.  Abel  and  Claussen  found,  for  example, 
in  14  out  of  17  persons  belonging  to  the  families  of  7 
cholera  patients,  cholera  vibrios,  in  some  of  them  for  a 
period  of  fourteen  days.  In  Hamburg  there  were  28 
such  cases  of  healthy  choleraic  individuals  with  abso- 
lutely normal  stools.  It  is  evident,  therefore,  that  an 
individual  susceptibility  is  requisite  to  produce  the  dis- 
ease. In  the  normal  healthy  stomach  the  hydrochloric 
acid  of  the  gastric  secretions  may  destroy  the  spirilla; 
and,  finally,  the  normal  vital  resistance  of  the  tissue 
cells  to  the  action  of  the  cholera  poison  may  be  taken 
into  consideration.  According  to  the  greater  or  less 
power  of  this  vital  resistance  of  the  body  the  same 
infectious  matter  may  give  rise  to  no  disturbance  what- 
ever, a  slight  diarrhoea,  or  it  may  lead  to  serious  results. 
Furthermore,  it  may  be  accepted  as  an  established  fact, 
that  recovery  from  one  attack  of  cholera  produces  per- 
sonal immunity  to  a  second  attack  for  a  considerable 
length  of  time.  This  does  not  appear  to  depend  upon 


SPIRILLUM  CHOLERA  ASIATICS.  585 

the  severity  of  the  attack;  for  cases  are  recorded  of 
persons  who  were  apparently  not  sick  at  all,  and  yet 
in  whom  an  acquired  immunity  was  produced.  How 
long  this  immunity  lasts  is  not  positively  known,  but 
probably  for  a  month  or  more,  so  that  the  same  person 
is  not  likely  to  be  taken  ill  again  with  cholera  during 
an  epidemic. 

Within  the  last  few  years  Haffkine,  in  India,  has 
succeeded  in  producing  an  artificial  immunity  against 
cholera  infection  by  means  of  subcutaneous  injections  of 
cholera  cultures.  In  over  200,000  persons  whom  he 
has  inoculated  the  results  obtained  would  undoubtedly 
seem  to  show  a  distinct  protective  influence  in  the  pre- 
ventive inoculations.  And  Kolle  has  found  that  the 
blood-serum  of  persons  inoculated  by  Haffkine' s  method 
gave  a  similar  reaction  to  that  of  persons  who  had  re- 
covered from  cholera. 

On  the  other  hand,  we  may  take  it  for  granted  that 
susceptibility  to  cholera  may  be  acquired  or  increased. 
For  instance,  there  is  no  doubt  that  gastric  and  intes- 
tinal disorders  produced  by  overeating,  etc.,  may  act 
as  contributing  causes  to  the  disease.  Other  predispos- 
ing causes  are  general  debility  from  poverty,  hunger, 
disease,  etc. 

Varieties  and  Variations  of  the  Cholera  Bacillus.  Cun- 
ningham, as  a  result  of  researches  made  in  Calcutta 
(1891),  arrives  at  the  conclusion  that  Koch's  comma 
bacillus  cannot  be  accepted  as  the  specific  etiological 
agent  in  this  disease:  First,  as  in  many  undoubted  cases 
of  cholera  he  has  failed  to  find  comma  bacilli;  second, 
because  in  one  case  he  found  three  different  species; 
and,  third,  because  in  one  case  the  indol  reaction  could 
not  be  obtained.  Since  Cunningham's  investigations 


586  BACTERIOLOGY. 

many  other  observers  have  reported  finding  varieties  of 
the  comma  bacillus.  Only  a  few  of  these  can  be  here 
mentioned,  of  which  there  is  any  certainty  that  they 
were  derived  from  true  cholera  cases.  Thus  Friedreich 
has  accurately  described  and  photographed  a  series  of 
forms  which,  however,  vary  but  little  from  the  original 
type.  But  more  interesting  than  the  reports  of  varie- 
ties are  the  observations  of  the  variability  of  the  cholera 
bacillus.  Claussen,  in  Esmarch'  s  institute,  isolated  from 
fresh  cholera  stools  vibrios  which  presented  in  plate 
cultures  a  different  appearance  of  the  colonies,  which 
showed  a  tendency  to  disintegrate  and  having  an  irreg- 
ular border.  The  nitrosoindol  reaction  was  absent; 
bouillon  cultures  were  non-pathogenic  to  guinea-pigs, 
and  stick  cultures  grew  slowly  and  uncharacteristically. 
On  repeated  inoculation,  however,  a  guinea-pig  died 
after  the  injection  of  1  c.c.  of  a  bouillon  culture;  in 
the  peritoneal  exudate  and  even  in  the  blood  character- 
istic bacilli  were  found  and  the  cultures  gave  the  indol 
reaction.  Celli  and  Santori,  in  Rome  (1893),  isolated 
from  the  stools  of  many  typical  cholera  cases  a  vibrio 
which  they  called  vibrio  romanus,  which  was  non-patho- 
genic for  animals,  gave  no  indol  reaction,  did  not  coag- 
ulate milk,  and  at  37°  C.  grew  neither  in  bouillon  nor 
on  agar.  After  cultivation  for  nine  months  it  gave  the 
indol  reaction  and  grew  at  37°  C.,  but  was  still  almost 
non-pathogenic.  Bordoni-Uffreduzzi  and  Abb  culti- 
vated from  a  typical  cholera  case  a  short  vibrio  which 
liquefied  gelatin  very  rapidly  and  presented  an  abnor- 
mal growth,  and  gave  a  yellow  growth  on  potato,  but 
which  on  continued  cultivation  became  more  and  more 
like  the  cholera  spirillum.  Variations  even  greater  than 
occur  in  these  varieties  of  cholera  spirilla  are  met  with 
among  diphtheria  bacilli. 


SPIRILLUM  CHOLERJE  ASIATICS.  587 

Plan  of  Procedure  for  the  Biological  Diagnosis  of  the 
Cholera  Vibrio.  A.  Dejecta  (fluid)  or  intestinal  con- 
tents of  a  cholera  patient  or  cholera  suspect. 

1.  Use  one  drop  (one  platinum  loop)  for  gelatin  plate- 
cultures,  making  two  dilutions.     Do  this  in  duplicate 
or  triplicate.     Cultivate  at  22°  C. 

2.  Inoculate  a  couple  of  bouillon  tubes  and  a  couple 
of  Dunham's  1  per  cent,  peptone  solution  with  one  drop 
each,  and  place  them  in  the  incubator  (37°  to  38°  C.) 
for  six  to  eight  hours. 

3.  Examine  a  drop  of  the  dejecta  in  the  hanging  drop. 

4.  Examine  a  drop  of  the  dejecta  in  stained  cover- 
glass  preparation.1 

5.  Make  gelatin  plates  from  one  drop  taken  from 
the  surface  of  each  of  the  bouillon  and  peptone  solution 
tubes  and  cultivate  at  22°  C. 

6.  As  soon  as  the  plates  (see  1  and  5)  are  sufficiently 
developed    (thirty-six   to   forty-eight   hours)  fish   the 
suspected  cholera  colonies  and  use  the  material  for  the 
following  procedures  : 

7.  Inoculate  six  or  eight  peptone  tubes  (1  per  cent, 
peptone,  0.5   per  cent.  NaCl  in  distilled  water)  and 
place  them  at  once  in  the  incubator.     Note  the  time. 

8.  Examine  hanging  drop  for  form,  size,  and  motility 
(and  arrangement). 

9.  Make  stained  cover-glass  preparations  and  exam- 
ine. 

1  These  direct  microscopical  examinations  of  the  intestinal  contents  are,  as 
a  rule,  very  unsatisfactory,  at  least  in  those  in  which  the  symptoms  are  not 
marked.  In  a  few  the  spirals  will  make  up  from  50  to  100  per  cent,  of  the 
bacteria  present.  In  most  of  the  cases  during  the  last  epidemic  in  New  York 
Dunham  found  abundance  of  columnar  epithelium  from  the  intestinal  mucous 
membrane,  numerous  straight,  thick  bacilli,  and  only  a  few  curved  bacilli  or 
segments  of  spirals— too  few  to  identify.  Plate  cultures  from  these  showed 
from  20  to  80  per  cent,  of  all  the  colonies  developing  to  be  cholera  spirilla. 


588  BACTERIOLOGY.. 

10.  Then  try  indol  reacton  with  the  same  tubes. 

11.  While  these  tubes  are   incubating  use  material 
from  the  suspected  colonies  on  the  plates  (1  and  5)  for 
hanging  drop  cultures. 

12.  Meanwhile  make  stained  cover-glass  prepara- 
tions from  other  colonies  of  suspected  cholera  on  the 
plates  (1  and  5). 

13.  Make  gelatin  tube  cultures   from  colonies  on 
plates  (1  and  5). 

14.  Make  gelatin  tube  cultures  daily  for  five  or  six 
days,  to  study  shape  of  growth  along  the  line  of  punc- 
ture to  preserve  the  culture. 

B.  Suspected  water. 

Add  to  500  c.c.  or  1  litre  of  the  water  to  be  exam- 
ined in  a  flask  half -full  enough  peptone-salt  solution 
(20  per  cent,  peptone  and  10  per  cent.  NaCl)  to  make 
a  1  per  cent,  solution  of  peptone.  Then  proceed  as  in  A. 

PFEIFFER'S  SERUM  REACTION.  All  authors  now 
agree  that  the  differentiation  of  the  cholera  vibrio  from 
other  similar  vibrios  cannot  always  be  made  by  the  cul- 
tural method,  nor  is  the  usual  inoculation  of  animals 
sufficient.  For  this  purpose  serum  is  employed  either 
by  making  intraperitoneal  injections  of  a  surely  fatal 
dose  of  the  suspected  spirillum  along  with  the  serum 
of  animals  immunized  to  undoubted  cholera  cultures, 
or  by  watching  the  action  of  the  spirillum  in  the  hang- 
ing drop  when  added  to  a  dilution  of  the  above  men- 
tioned serum,  so  as  to  note  whether  immobilization 
and  clumping  occurred. 


CHAPTER  XXXI Y. 

SPIRILLA    RESEMBLING   THAT   OF   CHOLERA — THE 
SPIRILLUM    OF   RELAPSING    FEVER. 

SPIRILLUM   OF  FINKLER  AND  PRIOR. 

FINKLER  and  Prior,  in  1884,  obtained  from  the  feces 
of  patients  with  cholera  nostras,  after  allowing  the 
dejecta  to  stand  for  some  days,  a  sprillum  which  is 
of  interest  mainly  because  it  simulates  the  comma 
bacillus  of  Koch,  but  differs  from  it  in  several  cultural 
peculiarities. 

Morphology.  More  or  less  curved  rods  with  an  aver- 
age length  of  2.4/z  and  a  breadth  of  0.4  to  0.6//,  some- 
what longer  and  thicker  than  the  spirillum  of  Asiatic 
cholera  and  not  so  uniform  in  diameter,  the  central  por- 
tion being  usually  wider  than  the  pointed  ends;  forms 
sometimes  S-shaped  and  spiral  filaments,  which  are  not 
as  numerous,  and  are  usually  shorter  than  those  formed 
by  the  cholera  spirillum.  Examined  in  the  hanging 
drop  they  are  seen  to  be  actively  motile.  A  single 
flagellum  is  attached  to  one  end  of  the  curved  segments. 
In  unfavorable  media  involution  forms  are  common. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters.  An  aerobic  and  facultative 
anaerobic,  liquefying  spirillum.  Does  not  form  spores. 
Upon  gelatin  plates  small,  white,  punctiforrn  colonies 
are  developed  at  the  end  of  twenty-four  hours,  which 


590 


BACTERIOLOGY. 


under  the  microscope  are  seen  to  be  finely  granular  and 
yellowish  or  yellowish-brown  in  color;  the  colonies  are 
round  with  more  sharply  denned  border,  less  coarsely 
granular  and  darker  in  color  than  those  of  the  comma 
bacillus.  Liquefaction  of  the  gelatin  around  these  col- 
onies progresses  rapidly,  and  at  the  end  of  forty-eight 
hours  is  usually  complete  in  plates  where  they  are  numer- 
The  surface  colonies  sink  quickly  into  the  gelatin 


ous. 


FIG.  80. 


Spirillum  of  Finkler  and  Prior.    X  1100  diameters. 

and  present  a  darker  peripheral  zone.  The  differenti- 
ation between  the  Finkler  and  Prior  and  cholera  spirilla 
can  readily  be  made  in  the  earlier  stages  of  their  growth. 
Later  on,  and  especially  when  the  cholera  colonies  are 
the  older,  the  diagnosis  is  not  so  easy.  In  gelatin  stick 
cultures  liquefaction  progresses  much  more  rapidly  than 
in  similar  cultures  of  the  cholera  spirillum,  and  a  stock- 
ing-shaped pouch  of  liquefied  gelatin  is  already  seen 
after  forty-eight  hours,  which  is  filled  with  a  cloudy 
liquid.  There  is  no  bubble  formation.  The  liquefac- 


SPIRILLUM  OF  FINKLER  AND  PRIOR.     591 

tion  increases,  and  in  twenty-four  hours  more  reaches 
the  sides  of  the  tube  in  the  upper  part  of  the  medium ; 
by  the  end  of  the  week  the  gelatin  is  usually  completely 
liquefied.  Upon  the  surface  of  the  liquefied  medium  a 
whitish  film  is  seen.  Upon  agar  there  is  a  somewhat 
more  luxuriant  growth  than  with  the  cholera  vibrio;  a 
slimy,  whitish-yellow  layer  covering  the  entire  surface 
is  quickly  developed.  Upon  potato  this  spirillum  grows 
at  the  room-temperature  and  produces  a  slimy,  grayish- 
yellow,  glistening  layer  which  soon  extends  over  the 
entire  surface.  The  cholera  spirillum  does  not  grow  at 
room-temperature,  and  in  the  incubator  produces  a  thin, 
brownish  layer.  Cultures  of  the  Finkler  and  Prior 
spirillum  give  off  a  strong  putrefactive  odor;  in  media 
containing  sugar,  according  to  Buchner,  an  acid  reac- 
tion is  produced  as  a  result  of  their  growth;  they  do 
not  form  indol  in  peptone  solutions;  and  they  have  a 
greater  resistance  to  desiccation  than  the  cholera  spiril- 
lum. The  absence  of  agglutination  with  a  dilution  of 
the  serum  of  an  animal  immunized  to  the  cholera 
spirillum  is  a  valuable  differential  sign. 

Pathogenesis.  When  injected  into  the  stomach  of 
guinea-pigs,  after  previous  injection  of  a  soda  solution 
and  opium,  the  Finkler  and  Prior  spirillum  is  some- 
what pathogenic  for  these  animals;  but  a  smaller  pro- 
portion die  from  such  injections  than  from  those  of 
the  fresh  cultures  of  cholera.  At  the  autopsy  the  in- 
testine is  pale,  and  its  watery  contents,  which  contain 
the  spirilla  in  great  numbers,  have  a  putrefactive  odor. 

This  organism  has  been  found  in  the  dejections  of 
some  healthy  persons  and  of  persons  affected  with  diar- 
rhoea or  cholera  nostras.  It  does  not  seem  to  have  any 
etiological  relation,  however,  with  this  disease  in  man, 


592  BACTERIOLOGY. 

as  since  its  disco  verey,  though  repeatedly  sought  for,  it 
has  seldom  been  found  by  subsequent  investigators. 

In  1884,  Miller  observed  a  curved  bacillus  in  a  hollow 
tooth,  which  from  its  behavior  in  microscopical  prepa- 
rations, in  cultures  and  animal  experiments,  is  probably 
identical  with  the  Finkler  and  Prior  spirillum;  and 
other  very  similar  spirilla  have  been  found  by  others. 

DENEKE'S  CHEESE  SPIRILLUM. 

Obtained  by  Deneke  (1885)  from  old  cheese,  but  since 
then  rarely  met  with.  Morphologically  and  culturally 
it  shows  greater  similarity  to  Koch's  comma  bacillus 
than  the  Finkler  and  Prior  spirillum,  but  can  be  read- 
ily differentiated  from  it  also. 

Morphology.  Curved  rods  and  long  spiral  filaments, 
somewhat  more  slender  than  the  cholera  spirillum,  the 
turns  in  the  spiral  threads  being  lower  and  closer 
together.  Has  a  single  flagelluin  attached  to  one  end. 

Stains  with  the  usual  aniline  colors. 

Biological  Characters.  An  aerobic  and  facultative 
anaerobic,  liquefying,  motile  spirillum.  Does  not  form 
spores.  Upon  gelatin  plates  small,  punctiform  colonies 
are  formed  at  the  end  of  twenty-four  hours,  which  when 
slightly  magnified  are  seen  to  be  circular  in  shape,  with 
sharply  defined  border  and  of  a  greenish-brown  color 
in  the  centre  and  paler  toward  the  margins.  Later, 
when  liquefaction  has  commenced,  the  sharp  contour  is 
often  lost.  The  liquefaction  progresses  more  rapidly 
than  with  the  cholera  bacillus,  but  not  so  energetically 
as  with  the  spirillum  of  Finkler  and  Prior.  In  gelatin 
stick  cultures  after  forty-eight  hours  a  stocking-like 
pouch  is  developed,  the  spirilla  sinking  to  the  bottom 


SPIRILL  UM  METSCHNIKO  VI.  593 

of  the  liquefied  gelatin  in  the  form  of  a  coiled  mass, 
while  a  thin,  yellowish  layer  forms  upon  the  surface ; 
complete  liquefaction  usually  occurs  in  about  two  weeks. 
Upon  the  surface  of  agar  a  thin,  yellowish  layer  is  de- 
veloped. Blood-serum  is  rapidly  liquefied.  The  indol 
reaction  in  peptone  solutions  is  absent. 

Pathogenesis.  Somewhat  pathogenic  for  guinea-pigs 
when  inoculated  by  Koch's  method  with  previous  ad- 
ministration of  soda  solution  and  laudanum. 

It  is  probable  that  this  organism,  from  the  locality  in 
which  it  is  found  and  its  behavior,  is  a  saprophyte. 

SPIRILLUM  METSCHNIKOVI. 

Discovered  in  1888,  in  Odessa,  by  Gamalei'a  in  the 
intestinal  contents  of  fowls  dying  of  an  infectious  dis- 
ease which  prevails  in  certain  parts  of  Russia  during 
the  summer  months,  and  which  presents  symptoms 
resembling  fowl-cholera.  GamaleiVs  experiments  show 
that  this  organism  is  the  cause  of  the  disease  mentioned. 
It  has  since  been  found  by  Pf  uhl  and  Pfeiffer  in  the  water 
of  the  Spree  at  Berlin,  and  in  the  Lahn  by  Kutchler. 

Morphology.  Morphologically  this  spirillum  is  almost 
identical  with  the  cholera  spirillum;  it  forms  curved 
rods  with  rounded  ends  and  spiral  filaments,  the  curved 
segments  being  somewhat  thicker,  shorter,  and  often 
more  decidedly  curved  than  the  comma  bacillus.  In 
the  blood  of  inoculated  pigeons  the  diameter  is  some 
times  twice  as  great  as  that  of  the  cholera  spirillum, 
and  almost  coccus-like  forms  are  often  found.  A  single, 
long,  undulating  flagellum  is  attached  to  one  end  of  the 
spiral  filaments  or  curved  rods.  In  old  cultures  beau- 
tiful long  spiral  filaments  may  be  seen. 

38 


594  BACTERIOLOGY. 

Stains  with  the  usual  aniline  colors,  but  not  by 
Gram's  method. 

Biological  Characters.  An  aerobic,  liquefying,  motile 
spirillum.  Upon  gelatin  plates  the  vibrio  Metschnikovi 
grows  considerably  faster  than  the  cholera  vibrio;  small, 
white  punctiform  colonies  are  developed  at  the  end  of 
twelve  hours;  these  rapidly  increase  in  size  and  cause 
liquefaction  of  the  gelatin  within  twenty-four  to  thirty 
hours.  At  the  end  of  three  days  large,  saucer-like 
areas  of  liquefaction  may  be  seen,  the  contents  of  which 
are  turbid,  as  a  rule.  Under  the  microscope  the 
colonies  appear  as  yellowish-brown  granular  masses, 
which  are  in  active  movement,  and  the  margins  are 
surrounded  by  a  border  of  radiating  filaments.  In 
gelatin  stick  cultures  the  growth  is  almost  twice  as  rapid 
as  the  cholera  bacillus.  In  bouillon  at  37°  C.  devel- 
opment is  very  rapid,  and  the  liquid  becomes  clouded 
and  opaque,  and  a  thin,  wrinkled  film  forms  upon  the 
surface.  On  the  addition  of  pure  sulphuric  acid  to 
twenty-four-hour  peptone  cultures  a  distinct  nitroso- 
indol  reaction  is  produced.  Milk  is  coagulated  and 
acquires  a  strongly  acid  reaction.  The  spirillum  does 
not  clump  and  lose  its  motility  with  the  diluted  serum 
from  an  animal  immunized  to  cholera. 

Pathogenesis.  The  vibrio  Metschnikovi  is  pathogenic 
for  fowls,  pigeons,  and  guiaea-pigs.  A  small  quantity 
of  a  culture  injected  into  the  breast  muscles  of  chickens 
and  pigeons  causes  their  death  with  the  local  and  gen- 
eral symptoms  of  fowl  cholera.  At  the  autopsy  the 
most  constant  appearance  is  hypersemia  of  the  entire 
alimentary  canal.  A  grayish-yellow  liquid,  more  or 
less  mixed  with  blood,  is  found  in  considerable  quan  • 
tity  in  the  small  intestine;  the  spleen  is  not  enlarged, 


SPIRILL  UM  METSCHNIKO  VI.  595 

rather  diminished  in  size,  and  the  organs  generally  are 
normal  in  appearance.  In  the  watery  fluid  large  num- 
bers of  spirilla  are  found;  they  are  found  in  the  blood 
of  pigeons  always,  but  only  in  the  blood  of  young  fowls. 
A  few  drops  of  a  pure  culture  inoculated  subcutane- 
ously  in  pigeons  cause  their  death  in  eight  to  twelve 
hours.  According  to  Gamale'ia,  fowls  may  be  infected 
by  giving  them  food  contaminated  with  the  cultures  of 
the  spirillum,  but  pigeons  resist  infection  in  this  way. 
Infection  may  also  be  produced  by  way  of  the  mouth 
by  Koch's  method,  a  solution  of  carbonate  of  soda  and 
laudanum  having  been  previously  administered.  The 
animals  then  die  with  symptoms  of  acute  gastro- 
enteritis; the  intestines  are  found  to  be  highly  inflamed 
and  their  liquid  contents  contain  numerous  spirilla. 
In  contradistinction  to  the  pathogenic  virulence  of 
these  spirilla  for  pigeons  and  guinea-pigs,  the  cholera 
spirillum  is  much  less  pathogenic.  Pigeons  are  not 
killed  by  the  intramuscular  inoculation  of  pure  fresh 
cultures  of  the  vibrio  cholerse.  Gamale'ia  has  claimed 
that  by  passing  the  cholera  spirillum  of  Koch  through 
a  series  of  pigeons,  by  successive  inoculation,  its  path- 
ogenic power  is  greatly  increased,  and  that  when  steril- 
ized cultures  of  this  virulent  variety  of  the  comma 
bacillus  are  injected  into  pigeons  they  become  immune 
against  the  pathogenic  action  of  the  vibrio  Metschni- 
kovi,  and  the  reverse.  But  Pfeiffer  has  shown  that 
this  statement  is  not  founded  upon  fact.  The  patho- 
genic action  of  the  vibrio  Metschnikovi  upon  pigeons 
and  guinea-pigs,  producing  in  these  animals  general 
septicsemia  and  death,  is,  therefore,  a  characteristic  point 
of  difference  between  this  and  the  spirillum  of  Asiatic 
cholera. 


596  BACTERIOLOGY. 

Within  recent  years  numerous  other  vibrios,  the  so- 
called  "  water  vibrios,"  have  been  found  while  looking 
for  the  cholera  bacillus,  the  identity  or  variation  of 
which  from  the  spirillum  of  cholera  it  has  been  ex- 
tremely difficult  to  determine,  as  morphological,  biolog- 
ical, and  pathogenical  examinations  have  led  to  no  posi- 
tive results. 

SPIRILLUM  OBERMEIERI  (Spirillum  of  Relapsing  Fever). 

First  observed  by  Obermeier  (1873)  in  the  blood  of 
persons  suffering  from  relapsing  fever. 

Morphology.  Long,  slender,  flexible,  spiral,  or  wavy 
filaments,  with  pointed  ends,  from  16//to40//  in  length 
and  from  one-quarter  to  one-third  the  thickness  of  the 
cholera  spirillum. 

Stains  readily  with  the  ordinary  aniline  colors,  es- 
pecially with  fuchsin,  Loffler's  solution  of  methylene- 
blue  and  Bismarck-brown.  Does  not  stain  by  Gram's 
method. 

Biological  Characters.  A  motile  spirillum  which 
has  not  been  cultivated  in  artificial  media.  Spore 
formation  has  not  been  demonstrated.  In  fresh  prep- 
arations from  the  blood  the  spirillum  exhibits  active 
progressive  movements  accompanied  by  very  rapid 
rotation  in  the  long  axis  of  the  spiral  filaments  or  by 
undulating  movements.  The  spirilla  are  found  exclu- 
sively in  the  blood  and  spleen  of  persons  suffering  from 
relapsing  fever,  never  in  the  secretions,  and  only  during 
the  fever,  not  in  the  intermissions,  or  at  most  singly  at 
the  beginning  of  an  attack.  When  preserved  in  blood- 
serum  or  a  0. 5  per  cent,  solution  of  salt  they  continue 
to  exhibit  active  movements  for  a  considerable  time. 


SPIRILLUM  OBERMEIERI.  597 

Efforts  to  cultivate  this  spirillum  in  artificial  culture 
media  have  thus  far  been  unsuccessful,  although  Koch 
has  observed  an  increase  in  the  length  of  the  spirilla 
and  the  formation  of  a  tangled  mass  of  filaments. 

Pathogenesis.  Inoculation  experiments  have  been  suc- 
cessfully made  on  man  and  monkeys.  Monkeys  when 
inoculated  with  human  blood  containing  the  spirilla 
take  sick  after  about  three  and  a  half  days,  but  show 
only  the  initial  febrile  attack;  no  relapses,  such  as  are 
characteristic  of  the  disease  in  man.  The  organisms 
are  found  in  the  blood,  and  at  the  height  of  the  fever 
in  the  other  organs  on  autopsy.  Extirpation  of  the 
spleen  renders  the  disease  more  dangerous  for  monkeys. 

Blood  from  one  animal,  taken  during  the  attack,  in- 
duces a  similar  febrile  paroxysm  when  inoculated  in 
another  monkey.  One  attack  does  not  preserve  the 
animal  experimented  on  from  a  second  attack  (Koch 
and  Carter). 

Very  little  is  known  bacteriologically  of  this  disease, 
but  from  the  fact  that  these  peculiarly  shaped  organ- 
isms are  constantly  and  exclusively  found  in  relapsing 
fever,  and  that  the  disease  can  be  transmitted  to  mon- 
keys by  inoculating  them  with  the  blood  containing  the 
spirilla,  it  may  be  assumed  that  they  are  the  true  cause 
of  the  disease. 


CHAPTER  XXXV. 

GLANDERS   BACILLUS. 

BACILLUS  MALLEI  (Bacillus  of  Glanders). 

THIS  bacillus  was  discovered  and  proved  to  be  the 
cause  of  glanders  by  isolation  in  pure  culture  and  com- 
munication to  animals  by  inoculation,  by  several  bacte- 
riologists almost  at  the  same  time  (1882),  viz.,  by  the 
investigations  of  Loftier,  Schiitz,  Israel,  Bouchard, 
Charrin,  Weichselbaum,  Kauzfeld,  and  Kitt.  It  is 
found  in  the  recent  nodules  in  animals  affected  with 
glanders,  and  in  the  discharge  from  the  nostrils,  pus 
from  the  specific  ulcers,  etc.,  and  occasionally  in  the 
blood. 

Morphology.  Small  bacilli  with  rounded  or  pointed 
ends,  from  0.25/*  to  0.4^  broad  and  from  1.5//  to  3// 
long;  usually  single,  but  sometimes  united  in  pairs,  or 
growing  out  to  long  filaments,  especially  in  potato  cul- 
tures. Frequently  breaks  up  into  short,  almost  coccus- 
like  elements  (Fig.  81). 

The  bacillus  mallei  stains  with  difficulty  with  the 
aniline  colors,  best  when  the  aqueous  solutions  of  these 
dyes  are  made  feebly  alkaline;  it  is  decolorized  by 
Gram's  method.  This  bacillus  presents  the  peculiarity 
of  losing  very  quickly  in  decolorizing  solutions  the  color 
imparted  to  it  by  the  aniline  staining  solutions.  For 
this  reason  it  is  difficult  to  stain  in  sections.  Loffier 
recommends  his  alkaline  methylene-blue  solution  for 


GLANDERS  BACILLUS.  599 

staining  sections,  and  for  decolorizing  a  mixture  con- 
taining 10  c.c.  of  distilled  water,  2  drops  of  strong 
sulphuric  acid,,  and  1  drop  of  a  5  per  cent,  solution  of 
oxalic  acid;  thin  sections  to  be  left  in  this  acid  solution 
for  five  seconds. 

FIG.  81. 


Glanders  bacilli.    Agar  culture.   X  1000  diameters. 

Biological  Characters.  An  aerobic,  non-motile  bacil- 
lus, whose  molecular  movements  are  so  active  that  they 
have  often  been  taken  for  motility.  It  grows  on  vari- 
ous culture  media  at  37°  C.  Development  takes  place 
slowly  at  22°  C.  and  ceases  at  43°  C.  It  does  not  form 
spores.  Exposure  for  ten  minutes  to  a  temperature  of 
55°  C.,  or  for  five  minutes  to  a  3  to  5  per  cent,  solution 
of  carbolic  acid,  or  for  two  minutes  to  a  1  : 5000  solu- 
tion of  mercuric  chloride,  was  effectual  in  destroying 
its  vitality.  As  a  rule,  the  bacilli  do  not  grow  after 
having  been  preserved  in  a  desiccated  condition  for  a 
week  or  two;  in  distilled  water  they  are  also  quickly 
destroyed.  The  bacillus  does  not  grow  in  infusions  of 


BA  CTERIOL  OOY. 

hay,  straw,  or  horse-manure,  and  it  is  doubtful  whether 
it  finds  conditions  in  nature  favorable  to  a  saprophytic 
existence.  It  grows  well  in  the  incubating  oven  on 
glycerin-agar.  Upon  this  medium  at  the  end  of  twenty- 
four  to  forty-eight  hours,  whitish,  transparent  colonies 
are  developed,  which  in  six  or  seven  days  may  attain  a 
diameter  of  7  or  8  mm.  On  blood-serum  a  moist, 
opaque,  slimy  layer  develops,  which  is  of  a  yellowish- 
brown  tinge.  The  growth  on  cooked  potato  is  especially 
characteristic.  At  the  end  of  twenty-four  to  thirty-six 
hours  at  37°  C.  a  moist,  yellow,  transparent  layer  de- 
velops; this  later  becomes  deeper  in  color,  and  finally 
takes  on  a  reddish-brown  color,  and  the  potato  about  it 
acquires  a  greenish-yellow  tint.  In  bouillon  it  causes 
diffuse  clouding,  with  ultimately  the  formation  of  a 
more  or  less  ropy  tenacious  sediment.  Milk  is  coagu- 
lated with  the  production  of  acid.  It  grows  on  media 
possessing  an  acid  reaction,  and  both  with  and  without 
oxygen. 

Pathogenesis.  The  bacillus  of  glanders  is  pathogenic 
for  a  number  of  animals.  Among  those  which  are  most 
susceptible  are  horses,  asses,  guinea-pigs,  cats,  dogs, 
ferrets,  moles,  and  field  mice;  sheep,  goats,  swine,  rab- 
bits, white  mice,  and  house  mice  are  much  less  suscep- 
tible; cattle  are  immune.  Man  is  susceptible,  and  in- 
fection not  infrequently  terminates  fatally. 

When  pure  cultures  of  the  bacillus  mallei  are  injected 
into  horses  and  other  susceptible  animals  true  glanders 
is  produced.  The  disease  is  characterized  in  the  horse 
by  the  formation  of  ulcers  upon  the  nasal  mucous  mem- 
brane, which  have  irregular,  thickened  margins,  and 
secrete  a  thin,  virulent  mucus;  the  submaxillary  lym- 
phatic glands  become  enlarged  and  form  a  tumor,  which 


GLANDERS  BACILLUS.  601 

is  often  lobulated;  other  lymphatic  glands  become  in- 
flamed, and  some  of  them  suppurate  and  open  exter- 
nally, leaving  deep,  open  ulcers;  the  lungs  are  also 
involved,  and  the  breathing  becomes  rapid  and  irreg- 
ular. In  farcy,  which  is  a  more  chronic  form  of  the 
disease,  circumscribed  swellings,  varying  in  size  from 
a  pea  to  a  hazel-nut,  appear  on  different  parts  of  the 
body,  especially  where  the  skin  is  thinnest;  these  sup- 
purate and  leave  angry- looking  ulcers  with  ragged 
edges,  from  which  there  is  an  abundant  purulent  dis- 
charge. The  bacillus  of  glanders  can  easily  be  obtained 
in  pure  cultures  from  the  interior  of  suppurating  nod- 
ules and  glands  which  have  not  yet  opened  to  the  sur- 
face, and  the  same  material  will  give  successful  results 
when  inoculated  into  susceptible  animals;  but  the  dis- 
charge from  the  nostrils  or  from  an  open  ulcer  contains 
comparatively  few  bacilli,  and  these  being  associated 
with  other  bacteria  which  grow  more  readily  on  the 
culture  media  than  the  bacillus  mallei,  it  is  not  easy  to 
obtain  pure  cultures  by  the  plate  method  from  such 
material,  and  here  animals  are  useful. 

Of  test  animals  guinea-pigs  and  field-mice  are  the 
most  susceptible.  In  guinea-pigs  subcutaneous  injec- 
tions are  followed  in  four  or  five  days  by  swelling  at 
the  point  of  inoculation,  and  a  tumor  with  caseous  con- 
tents soon  develops;  then  ulceration  of  the  skin  takes 
place,  and  a  chronic  purulent  ulcer  is  formed.  The 
lymphatic  glands  become  inflamed  and  general  symp- 
toms of  infection  are  developed  in  from  two  to  four 
weeks;  the  glands  suppurate  and  in  males  the  testicles 
are  involved;  finally  purulent  inflammation  of  the  joints 
occur,  and  death  ensues  from  exhaustion.  The  forma- 
tion of  the  specific  ulcers  upon  the  nasal  mucous  mem- 


602  BA  CTER10L  OGY. 

brane,  which  characterize  the  disease  in  the  horse,  rarely 
result  from  inoculation  of  the  guinea-pig.  The  process 
is  often  prolonged,  or  it  remains  localized  on  the  skin. 
Guinea-pigs  succumb  more  rapidly  to  intraperitoneal 
injection,  usually  in  from  eight  to  ten  days,  and  in 
males  the  testicles  are  invariably  affected. 

Glanders  occurs  as  a  natural  infection  only  in  horses 
and  asses;  the  disease  is  occasionally  communicated  to 
man  by  contact  with  affected  animals,  and  usually  by 
inoculation  on  an  abraded  surface  of  the  skin.  The 
contagion  may  also  be  received  on  the  mucous  mem- 
brane. Infection  has  sometimes  been  produced  in 
bacteriological  laboratories.  In  the  horse,  the  disease 
may  be  localized  in  the  nose  (glanders)  or  beneath  the 
skin  (farcy).  The  essential  lesion  is  the  granulomatous 
tumor,  characterized  by  the  presence  of  numerous  lym- 
phoid  and  epithelioid  cells,  among  and  in  which  are  seen 
the  glanders  bacilli.  These  nodular  masses  tend  to  break 
down  rapidly,  and  on  the  mucous  membrane  form  ulcers, 
while  beneath  the  skin  they  form  abscesses.  The  glan- 
ders nodules  may  also  occur  in  the  internal  organs.  An 
acute  and  chronic  form  of  glanders  may  be  recognized 
in  man,  and  an  acute  and  a  chronic  form  of  farcy.  The 
disease  is  fatal  in  a  large  proportion  of  cases.  It  is 
transmissible  also  from  man  to  man.  Washer-women 
have  been  infected  from  the  clothes  of  a  patient.  The 
infective  material  exists  in  the  secretions  of  the  nose, 
in  the  pus  of  glanders  nodules,  and  sometimes  in  blood; 
it  may  occasionaly  be  found  in  the  secretions  of  healthy 
glands,  as  in  the  urine,  milk,  and  saliva,  and  also  in 
the  foetus  of  diseased  animals  (Bonome).  From  recent 
observations  it  appears  that  glanders  is  by  no  means 
an  uncommon  disease  among  horses,  particularly  in 


GLANDERS  BA  GILL  US.  603 

southern  countries,  sometimes  taking  a  mild  course  and 
remaining  latent  for  a  considerable  time  (Semmer  and 
Babes).  Apparently  healthy  horses,  therefore,  may  pos- 
sibly spread  the  disease. 

Attenuation  of  virulence  occurs  in  cultures  which 
have  been  kept  for  some  time,  and  inoculations  with 
such  cultures  may  give  a  negative  result;  or,  when  con- 
siderable quantities  are  injected,  may  produce  a  fatal 
result  at  a  later  date  than  is  usual  when  small  amounts 
of  a  recent  culture  are  injected  into  susceptible  animals. 

Several  attempts  have  been  made  by  investigators  to 
produce  artificial  immunity  against  glanders,  but  so  far 
with  unsatisfactory  results.  According  to  Strauss,  dogs 
may  be  protected  by  intravenous  inoculations  of  small 
quantities  of  living  bacilli  against  an  injection  with 
large  quantities  which  usually  kill  them.  Fenger  has 
found  that  animals  inoculated  with  glanders  bacilli 
react  less  powerfully  to  fresh  injections;  and  that  rab- 
bits which  have  recovered  from  an  injection  of  glanders 
are  subsequently  immune,  the  immunity  lasting  for  from 
three  to  six  weeks.  Ladowski  has  obtained  positive  re- 
sults also  in  rabbits  and  cats  by  intravenous  injections 
of  sterilized  cultures.  Other  observers  have  reported 
not  only  the  production  of  immunity,  but  also  cures,  by 
the  use  of  mallein.  Mallein  is  produced  by  evaporat- 
ing a  six-weeks7  old  culture  of  the  glanders  bacillus  in 
5  per  cent,  glycerin  nutrient  veal  bouillon  to  10  per 
cent,  of  its  original  bulk.  It  is  made  in  the  same  way 
as  Koch's  crude  tuberculin  from  the  tubercle  bacillus 
cultures.  j  %  . 

Differential  Diagnosis.  It  is  often  difficult  to  demon- 
strate microscopically  the  presence  of  the  bacillus  of 
glanders  in  the  nodules  which  have  undergone  purulent 


CHAPTER  XXXVI. 

BUBONIC   PLAGUE   BACILLUS — YELLOW    FEVER 
BACILLUS — WHOOPING-COUGH    BACILLUS. 

BACILLUS  OF  BUBONIC  PLAGUE  (Bacillus  Pestis 
Bubonicse  —  Kitasato ;  Bacterium  Pestis). 

DISCOVERED  simultaneously  by  Kitasato  and  Yersin 
(1894)  during  an  epidemic  of  the  bubonic  plague  in 
China.  It  is  found  in  large  numbers  in  the  pus  from 

FIG.  82. 


Bacillus  of  bubonic  plague.    X  1000  diameters. 

the  buboes  characteristic  of  this  disease  and  in  the 
lymphatic  glands;  more  rarely  in  the  internal  organs 
and  in  the  blood,  in  which  it  occurs  in  acute  hemor- 


BACILLUS  OF  BUBONIC  PLAGUE.  607 

rhagic  cases  and  shortly  before  death.  It  also  occurs 
in  the  feces  of  men  and  animals. 

Morphology.  Short  thick  rods,  with  rounded  ends, 
frequently  occurring  in  short  chains  and  often  sur- 
rounded by  a  capsule.  When  obtained  from  cultures 
the  bacilli  present  considerable  spherical  enlargement 
(Fig.  82). 

Stains  readily  with  the  ordinary  aniline  dyes,  the 
ends  being  usually  more  deeply  colored  than  the  central 
portion;  does  not  stain  by  Gram's  method. 

Biological  Characters.  An  aerobic,  non-motile  bacil- 
lus. Does  not  form  spores.  Grows  on  the  usual  culture 
media.  Does  not  liquefy  gelatin.  Grows  best  on  blood- 
serum  in  the  incubator,  the  growth  appearing  on  the 
surface  after  twenty-four  to  forty-eight  hours,  in  the 
form  of  white,  moist,  transparent  and  iridescent  col- 
onies. It  grows  rapidly  on  glycerin-agar,  forming  a 
grayish-white  surface  growth.  In  bouillon  a  very  char- 
acteristic appearance  is  produced,  the  culture  medium 
remaining  clear  while  a  granular  or  grumous  deposit 
forms  on  the  walls  and  on  the  bottom  of  the  tube. 

Pathogenesis.  This  bacillus  is  pathogenic  for  rats, 
mice,  guinea-pigs,  monkeys,  rabbits,  flies,  and  other 
insects,  which  usually  die  within  two  or  three  days 
after  inoculation.  Then  at  the  point  of  inoculation  is 
found  a  somewhat  hemorrhagic  infiltration  and  rcdema, 
with  enlargements  of  the  neighboring  lymph-glands, 
hemorrhages  into  the  peritoneal  cavity  and  parenchy- 
matous  congestion  of  the  organs.  The  spleen  sometimes 
shows  minute  nodules  resembling  miliary  tubercles. 
Microscopically  the  bacilli  are  found  in  all  the  organs 
and  in  the  blood.  The  disease  is  rapidly  communicated 
from  one  animal  to  another,  and  thus  its  extension  is 


CHAPTER  XXXVI. 

BUBONIC   PLAGUE   BACILLUS — YELLOW    FEVER 
BACILLUS — W HOOPING-COUG  H   B A CILLUS. 

BACILLUS  OF  BUBONIC  PLAGUE  (Bacillus  Pestis 
Bubonicae  — Kitasato ;  Bacterium  Pestis). 

DISCOVERED  simultaneously  by  Kitasato  and  Yersin 
(1894)  during  an  epidemic  of  the  bubonic  plague  in 
China.  It  is  found  in  large  numbers  in  the  pus  from 

FIG.  82. 


Bacillus  of  bubonic  plague.    X  1000  diameters. 

the  buboes  characteristic  of  this  disease  and  in  the 
lymphatic  glands;  more  rarely  in  the  internal  organs 
and  in  the  blood,  in  which  it  occurs  in  acute  hemor- 


BACILLUS  OF  BUBONIC  PLAQUE.  607 

rhagic  cases  and  shortly  before  death.  It  also  occurs 
in  the  feces  of  men  and  animals. 

Morphology.  Short  thick  rods,  with  rounded  ends, 
frequently  occurring  in  short  chains  and  often  sur- 
rounded by  a  capsule.  When  obtained  from  cultures 
the  bacilli  present  considerable  spherical  enlargement 
(Fig.  82). 

Stains  readily  with  the  ordinary  aniline  dyes,  the 
ends  being  usually  more  deeply  colored  than  the  central 
portion;  does  not  stain  by  Gram's  method. 

Biological  Characters.  An  aerobic,  non-motile  bacil- 
lus. Does  not  form  spores.  Grows  on  the  usual  culture 
media.  Does  not  liquefy  gelatin.  Grows  best  on  blood- 
serum  in  the  incubator,  the  growth  appearing  on  the 
surface  after  twenty-four  to  forty-eight  hours,  in  the 
form  of  white,  moist,  transparent  and  iridescent  col- 
onies. It  grows  rapidly  on  glycerin-agar,  forming  a 
grayish- white  surface  growth.  In  bouillon  a  very  char- 
acteristic appearance  is  produced,  the  culture  medium 
remaining  clear  while  a  granular  or  grumous  deposit 
forms  on  the  walls  and  on  the  bottom  of  the  tube. 

Pathogenesis.  This  bacillus  is  pathogenic  for  rats, 
mice,  guinea-pigs,  monkeys,  rabbits,  flies,  and  other 
insects,  which  usually  die  within  two  or  three  days 
after  inoculation.  Then  at  the  point  of  inoculation  is 
found  a  somewhat  hemorrhagic  infiltration  and  oedema, 
with  enlargements  of  the  neighboring  lymph-glands, 
hemorrhages  into  the  peritoneal  cavity  and  parenchy- 
matous  congestion  of  the  organs.  The  spleen  sometimes 
shows  minute  nodules  resembling  miliary  tubercles. 
Microscopically  the  bacilli  are  found  in  all  the  organs 
and  in  the  blood.  The  disease  is  rapidly  communicated 
from  one  animal  to  another,  and  thus  its  extension  is 


608  BACTERIOLOGY. 

facilitated.  During  epidemics,  rats,  mice,  and  flies,  in 
large  numbers,  become  infected  and  die,  and  the  disease 
is  apparently  transmitted  through  them  to  man.  The 
organism  is  found  in  the  feces  of  sick  animals,  in  the 
dust  of  infected  houses,  and  in  the  soil. 

The  virulence  of  the  bacilli  in  cultures  and  in  nature 
seems  to  vary  considerably,  and  is  rather  rapidly  lost 
when  grown  on  artificial  media.  The  growth  in  cul- 
tures becomes  more  abundant  after  frequent  transplan- 
tation. The  virulence  of  the  organism  is  increased  by 
successive  inoculation  in  certain  animal  species,  and 
then  its  pathogenic  properties  for  other  species  are  less 
marked. 

Yersin,  Calmette,  and  Borrel  have  succeeded  in  im- 
munizing animals  against  the  bacillus  of  bubonic  plague 
by  inoculation,  by  the  intravenous  or  intraperitoneal 
injection  of  dead  cultures,  or  by  repeated  subcutaneous 
inoculation.  They  also  succeeded  in  immunizing  rab- 
bits and  horses,  so  that  the  serum  afforded  protection 
to  small  animals,  after  subcutaneous  injection  of  viru- 
lent cultures,  and  even  cured  those  which  had  been 
inoculated,  if  administered  within  twelve  hours  after 
injection.  The  serum  has  considerable  antitoxic  as 
well  as  bactericidal  properties.  More  recently  this 
serum  has  been  applied  by  Yersin  to  the  treatment  of 
bubonic  plague  in  man,  with  very  promising  results. 
Experience  has  shown  that  the  treatment  is  more 
efficacious  the  earlier  the  stage  of  the  disease.  When 
treatment  is  begun  in  the  first  day  of  the  attack,  fever 
and  all  alarming  symptoms  usually  disappear  with 
astouishing  rapidity.  In  cases  treated  at  a  later  stage 
larger  doses  of  the  serum  are  required,  and  even  in  the 
favorable  cases  suppuration  of  the  buboes  is  not  always 


BACILLUS  ICTEROIDES.  609 

prevented.  In  some  of  the  early  cases  and  in  many  of 
the  rather  late  ones  the  serum  fails.  When  the  disease 
is  far  advanced  the  serum  is  powerless.  For  immu- 
nizing purposes  the  serum  should  be  valuable,  and  a 
single  injection  would  probably  give  protection  for 
several  weeks. 

Haffkine,  in  India,  has  recently  applied  his  method 
of  preventive  inoculation  to  the  bubonic  plague,  as  he 
previously  did  with  cholera  and  apparently  with  equally 
good  results.  This  method  consists  in  an  inoculation 
of  dead  cultures,  and  is  essentially  a  protective  rather 
than  a  curative  treatment.  It  gives  after  six  to  ten 
days  a  considerable  immunity,  lasting  a  month  or  more. 
By  means  of  these  two  methods  of  inoculation,  along 
with  strict  quarantine  regulations,  it  is  to  be  hoped 
that  this  disease  which  under  the  name  of  Black 
Death  once  decimated  the  populations  of  the  earth,  and 
which  in  the  East  still  causes  great  mortality  at  times, 
may  finally  be  greatly  restricted  or  even  stamped  out 
altogether. 

BACILLUS  ICTEROIDES  (Bacillus  of  Yellow  Fever). 

In  1897  Sanarelli  announced  the  discovery  of  a  micro- 
organism which  he  claimed  to  be  the  specific  cause  of 
yellow  fever.  This  he  called  the  "  bacillus  icteroides." 
It  is  found  in  the  circulating  blood  and  in  the  tissues  of 
yellow  fever  patients. 

Morphology.  Short  rods  with  rounded  extremities, 
single  or  united  in  pairs  in  cultures  and  in  groups  in 
the  tissues,  from  I/*  to  2//  in  length,  and  generally  two 
to  three  times  longer  than  broad.  Somewhat  polymor- 
phous. It  resembles  the  colon  bacilli. 

39 


610  BACTERIOLOGY. 

Stains  readily  with  the  ordinary  aniline  dyes,  but 
not  by  Gram's  method. 

Biological  Characters.  A  motile,  facultative  anaero- 
bic, non-liquefying  bacillus.  Does  not  form  spores  as 
far  as  known.  Grows  readily  in  all  the  ordinary  cul- 
ture media,  at  the  room-temperature,  but  best  at  37°  C. 
iu  the  incubator.  On  yelatin  plates  it  forms  rounded 
colonies,  transparent  and  granular  It  never  liquefies 
gelatin.  In  bouillon  the  bacillus  grows  quickly,  without 
forming  either  a  pellicle  or  deposit.  On  blood-serum 
its  growth  is  almost  imperceptible.  Cultures  on  agar 
are  characteristic,  according  to  Sanarelli.  When  the 
colonies  grow  in  the  incubator  they  present  an  appear- 
ance that  does  not  differ  from  many  other  species;  they 
are  rounded,  of  a  slightly  iridescent  gray  color,  trans- 
parent, even  in  surface,  and  regular  in  outline.  Grown 
at  the  room-temperature  from  20°  to  22°  C.,  they  ap- 
pear like  drops  of  milk,  opaque,  projecting,  and  with 
pearly  reflections,  completely  distinct  from  those  grown 
in  the  incubator.  These  different  modes  of  evolution 
Sanarelli  considers  to  be  an  important  diagnostic  point; 
first  exposing  the  cultures  for  from  twelve  to  sixteen 
hours  to  the  temperature  of  the  incubator,  and  after- 
ward for  twelve  to  sixteen  hours  more  to  the  tempera- 
ture of  the  air. 

The  bacillus  icteroides  ferments  glucose  and  sacch- 
arose, but  does  not  coagulate  milk;  produces  little 
indol,  and  is  quite  resistant  to  desiccation;  it  dies  in 
water  at  60°  C.,  or  after  being  exposed  for  seven  hours 
to  the  sunlight,  and  lives  for  a  long  time  in  sea-water. 

Pathogenesis.  It  is  pathogenic  for  the  greater  num- 
ber of  the  domestic  animals;  but  birds  are  completely 
refractory.  According  to  the  discoverer  the  dog  lends 


BA  GILL  US  ICTEROIDES.  61 1 

itself  particularly  to  experimentation  with  this  organ- 
ism. The  virus  should  be  injected  into  a  vein.  The 
lesions  found  after  death  are  said  to  be  almost  identical 
with  those  in  human  yellow  fever  cadavers.  There  is 
fatty  degeneration  of  the  liver  and  kidneys,  accompa- 
nied by  acute  parenchymatous  nephritis.  The  digestive 
apparatus  shows  lesions  of  hemorrhagic  gastro-enteritis. 
The  bacilli  are  found  in  the  blood  and  the  organs  in 
variable  quantity  and  in  a  state  of  absolute  purity;  at 
times,  they  may  be  associated  with  the  B.  coli  and  the 
streptococcus. 

The  disease  may  be  transmitted  experimentally  even 
by  the  respiratory  tract  to  rabbits  and  guinea-pigs;  the 
bacteriological  examination  of  these  cases  shows,  at 
least,  the  existence  of  toxic  processes  similar  with  that 
which  takes  place  in  man.  The  toxin  is  obtained  by 
filtering  twenty  to  twenty-five  days'  old  cultures  in 
broth.  It  withstands  a  temperature  of  70°  C.,  but  is 
sensibly  weakened  by  boiling. 

According  to  Sanarelli,  infection  in  the  human  sub- 
ject does  not  take  place  by  the  digestive  but  by  the 
respiratory  tract;  and  he  suggests  that  the  common 
moulds  of  the  atmosphere  may  constitute  protectors  of 
the  bacillus  icteroides. 

Sanarelli  has  also  prepared  a  protective  or  curative 
serum  for  the  treatment  of  the  disease,  which  he  calls 
"  anti-amarylic  serum."  This  serum  has  not  been 
sufficiently  tested  as  yet  to  form  any  definite  conclu- 
sions as  to  its  value ;  but  because  of  its  not  being  an 
antitoxin,  it  does  not  tend  to  overcome  the  toxins  of 
yellow  fever  produced  in  the  system,  and  depends  for 
its  curative  and  prophylactic  properties  upon  its  germi- 
cidal  influence.  Hence,  it  is  argued  by  Sanarelli  that 


612  BACTERIOLOGY. 

its  use  will  be  absolutely  negative  in  cases  in  which  an 
amount  of  toxin  has  been  produced  sufficient  to  destroy 
life.  He,  therefore,  insists  upon  the  early  use  of  the 
serum,  and  thus  the  destruction  of  the  organism  before 
it  has  elaborated  the  fatal  proportion  of  its  toxin.  It 
is  claimed  that  in  thirty-one  cases  treated  with  the  serum 
the  mortality  was  only  32  per  cent.,  whereas  in  South 
America, where  the  treatment  was  applied,  the  mortality 
of  yellow  fever  often  rises  to  50  per  cent.  (Sanarelli). 

Since  Sanarelli' s  supposed  discovery  a  number  of 
investigations  have  been  made  into  the  causal  relation  of 
this  organism  to  the  disease,  some  of  which  seem  to 
cast  considerable  doubt  upon  its  being  the  specific 
cause  of  yellow  fever,  while  others  are  in  its  favor. 
Novy,  in  a  recent  paper  (September,  1898),  comes  to 
the  conclusion,  after  an  exhaustive  examination  into 
this  subject,  that  the  Sanarelli  bacillus  belongs  to  the 
typhoid  group,  and  that  it  is  not  the  cause  of  yellow 
fever,  which  is  yet  to  be  worked  out. 

Novy's  chief  objection  to  this  bacillus  rests  upon 
the  fact  that  yellow  fever  is  stopped  by  a  frost  and  that 
this  bacillus  is  not  injured  by  much  greater  cold.  It  is 
perfectly  possible,  however,  that  the  infection  is  carried 
in  some  indirect  way,  as  by  insects,  and  that  the 
carriers  of  infection  are  affected  by  the  cold,  and  so  the 
dissemination  of  the  poison  is  prevented.  The  long 
immunity  conferred  by  an  attack  and  the  peculiar  effect 
of  cold  on  the  spread  of  the  disease  are  nevertheless 
difficult  to  explain  by  means  of  the  known  character- 
istics of  this  bacillus.  In  September  a  report  by 
Geddings  and  Wasdin  appeared  which  favored  the 
claims  of  Sanarelli,  they  finding  the  bacilli  in  almost 
every  case  of  yellow  fever,  and  not  in  any  which  were 


THE  BACILLUS  OF  WHOOPING-COUGH.     613 

not  yellow  fever.  According  to  them  the  lungs  were 
the  earliest  and  the  chief  seat  of  the  lesions.  Their 
report  adds  to  the  mystery  of  the  effect  of  cold  on 
stopping  the  spread  of  the  disease,  for  nearly  all  respi- 
ratory diseases  due  to  bacteria  tend  to  increase  in  cold 
weather,  and  certainly  their  spread  is  not  stopped  im- 
mediately. This  bacillus  can  as  yet  be  considered  as 
only  the  possible  cause  of  yellow  fever. 

THE  BACILLUS  OF  WHOOPING-COUGH. 

From  lime  to  time  observers  have  found  in  the 
sputum  of  persons  suffering  from  whooping-cough 
small  bacilli,  often  in  great  numbers.  These  have  been 
studied  lately  especially  by  Koplik1  and  Czaplewski2 
and  Hensel,  who  believe  that  these  bacilli  are  the  cause 
of  the  disease.  They  are  small  bacilli  of  about  the  size 
of  the  influenza  bacillus,  and  grow  on  blood-serum  and 
nutrient  agar  in  tiny  colonies.  Mice  and  rabbits  die 
after  intravenous  inoculations.  No  symptoms  similar 
to  those  in  man  are  noted.  The  observers  differ  as  to 
the  description  of  the  bacilli,  and  those  interested  are 
referred  to  the  original  articles.  The  examination  for 
these  bacilli,  if  they  prove  to  be  the  true  cause  of  the 
disease,  may  prove  of  diagnostic  importance,  and  also 
be  of  use  in  detecting  sources  of  contagion. 

i  Centralblatt  fur  Bact.  Abth.,  1  Bd.  22,  p.  222  *  Ibid.,  p.  641. 


APPENDIX. 


BRIEF   DESCRIPTIONS   OF   A  FEW   REPRESENTATIVE 

PATHOGENIC  MICRO-ORGANISMS  WHICH 

ARE  NOT  BACTERIA. 


CHAPTER   XXX VI T. 

THE   STREPTOTHKIX   GROUP — FAVU8   AND    RINGWORM 
FUNGI. 

THE  varieties  of  the  streptothrix  group  have  as  yet 
not  beeu  clearly  described.  Some  at  least  are  patho- 
genic. This  group  of  micro-organisms  while  having 
many  affinities  with  the  bacteria,  yet  differs  from  them 
in  many  important  respects  which  link  them  with  the 
fungi.  They  develop  from  spore-like  bodies  into 
cylindrical  dichotomously  branching  threads  which 
grow  into  colonies,  the  appearance  of  which  suggests  a 
mass  of  radiating  filaments.  Under  favorable  condi- 
tions certain  of  the  threads  become  fruit-hypse,  and 
these  break  up  into  chains  of  round  spore-like  bodies, 
which  do  not,  however,  have  the  same  staining  reac- 
tions nor  resisting  powers  as  true  spores.  The  tubercle 
grass  and  diphtheria  bacilli  are  by  some  believed  to 
properly  belong  in  the  streptothrix  group  on  account 
of  the  true  branching  forms  developed  by  them  under 
certain  conditions.  The  best  known  of  the  strepto- 
thrix group  is  the  actinomyces  fungus. 


APPENDIX. 


STREPTOTHRJX  ACTINOMYCES  (Actinomyces  Fungus  ; 
Ray  Fungus). 

This  micro-organism  was  first  described  by  Bollinger 
(1877)  in  the  ox,  in  which  it  forms  the  affection  known 
as  "  big-jaw.  "  In  man  the  disease  was  first  described 
by  J.  Israel  (1885),  and  subsequently  Ponfick  insisted 
upon  the  identity  of  the  disease  in  man  and  cattle.  To 
Bostrom  we  owe  the  most  elaborate  and  accurate  account 
of  the  structure  and  development  of  this  organism. 

Morphology.  In  both  man  and  animals  it  can  be  seen 
in  the  pus  from  the  affected  regions  as  small  yellowish 
granules  from  0.5  to  2  mm.  in  diameter.  Microscopi- 
cally these  bodies  are  seen  to  be  made  up  of  threads 
which  radiate  from  a  centre  and  present  bulbous,  club- 
like  terminations.  These  club  -like  terminations  are 
characteristic  of  the  actinomyces.  They  are  generally 
arranged  in  pairs,  closely  crowded  together,  and  are  very 
glistening  in  appearance.  The  threads  which  compose 
the  central  mass  of  the  granules  are  from  0.3//  to  0.5// 
in  diameter;  the  clubs  are  from  6//  to  8//  in  diameter. 

The  organism  is  stained  with  the  ordinary  aniline 
colors,  also  by  Gram's  solution;  when  stained  with 
gentian-  violet  and  by  Gram's  method  the  threads  ap- 
pear more  distinct  than  when  stained  with  methylene- 
blue. 

Biological  Characters.  It  grows  in  all  the  ordinary 
artificial  culture  media,  but  often  several  cultures  have 
to  be  made  before  getting  a  satisfactory  one.  It  de- 
velops at  the  room-temperature,  and  grows  both  with 
and  without  oxygen,  but  best  with  access  of  air  and  at 
the  temperature  of  the  body. 

Growth  on  Blood-serum  and  Agar.  Isolated  colonies 
at  first  develop  on  the  surface  of  these  media,  but  on 


STREPTOTHRIX  A CTINOMYCES.  617 

keeping  the  cultures  for  a  week  or  more  the  colonies 
run  together  and  form  a  thick,  wrinkled  mass  which 
sinks  into  the  media.  The  individual  colonies  are 
yellowish  to  red  in  color,  and  are  covered  by  a  whit- 
ish, fluffy  down,  consisting  of  cobweb-like  threads. 
On  touching  the  colonies  they  will  be  found  to  cling 
close  to  the  medium,  and  on  forcible  removal  they  go 
to  pieces.  On  making  a  smear  preparation  the  thread- 
like structure  will  be  seen.  In  stick  cultures  the  growth 
usually  presents  a  tree-like  appearance,  but  it  varies 
very  considerably;  there  may  be  no  reddish  pigmenta- 
tion, and  the  cobweb-like  threads  are  not  always  devel- 
oped on  the  surface.  Occasionally  the  culture  on  agar 
is  colored  brown. 

The  Growth  in  Bouillon.  When  the  medium  is  allowed 
to  stand  perfectly  still  a  distinct  granular  growth  occurs, 
but  on  agitation  these  grains  are  broken  up,  though  the 
liquid  is  never  clouded.  At  times  large  flakes  or  a  mem- 
branous film  form  on  the  surface  of  the  medium,  upon 
which  develops  the  fluffy  down  previously  described. 

The  Growth  on  Potato,  On  this  medium  the  growth 
is  somewhat  slower,  resulting  in  a  thick,  viscid,  mem- 
branous deposit  on  the  surface  of  the  potato  on  which 
the  same  cobweb-like  threads  are  developed.  The  color 
is  yellowish-red. 

The  cultures  are  quite  resistant  to  outside  influences; 
dried  they  may  be  kept  for  a  year  or  more ;  they  are 
killed  by  a  temperature  of  75°  C.,  the  time  of  exposure 
being  five  minutes. 

Occurrence  in  Animals.  Actinomycosis  is  quite  prev- 
alent among  cattle,  in  which  it  occurs  endemically; 
it  is  more  rare  among  swine  and  horses,  and  is  some- 
times found  in  man.  The  disease  is  probably  not  con- 


618  .    APPENDIX. 

tagious,  but  infection  may  result  from  the  ingestion  of 
vegetable  products  which  contain  the  fungus.  The 
cereal  grains,  which  from  their  nature  are  capable  of 
penetrating  the  tissues,  have  been  repeatedly  found  in 
centres  of  actinomycotic  infection.  This  usually  occurs 
in  the  vicinity  of  the  mouth,  where  injuries  have  been 
accidentally  caused.  The  micro-organism  may  also  be 
introduced  by  means  of  carious  teeth.  Cutaneous  in- 
fection has  been  produced  by  wood-splinters,  and  infec- 
tion of  the  lungs  by  aspiration  of  fragments  of  teeth 
containing  the  fungus.  The  further  distribution  of  the 
fungus  after  it  is  introduced  into  the  tissues  is  effected 
partly  by  its  growth  and  partly  by  conveyance  by 
means  of  the  lymphatics  and  leucocytes.  Not  infre- 
quently a  mixed  infection  with  the  pyogenic  cocci 
occurs  in  actinomycosis. 

In  the  earliest  stages  of  its  growth  the  parasite  gives 
rise  to  a  small  granulation  tumor,  not  unlike  that  pro- 
duced by  the  tubercle  bacillus,  which  contains,  in  addi- 
tion to  small  round  cells,  epithelial  elements  and  giant 
cells.  After  it  reaches  a  certain  size  there  is  great  pro- 
liferation of  the  surrounding  connective  tissue,  and  the 
growth  may,  particularly  in  the  jaw,  look  like,  and  was 
long  mistaken  for,  osteosarcoma.  Finally,  suppuration 
occurs,  which,  according  to  Israel,  may  be  produced 
directly  by  the  fungus  itself. 

The  experimental  production  of  actinomycosis  in  ani- 
mals has  been  followed  by  negative  or  very  unsatisfac- 
tory results.  When  artificially  introduced  into  the 
tissues  the  organism  is  either  absorbed  or  encapsulated. 
If  introduced  in  large  quantities  multiple  nodules  are 
apparently  formed  in  some  cases,  which  may  suggest 
the  production  of  a  general  infective  process;  but  on 
closer  inspection  of  these  nodules  the  thread-like  por- 


THE  FUNGI.  619 

tion  of  the  fungus  are  found  to  have  disappeared,  leav- 
ing only  the  remains  of  the  club-like  ends,  thus  showing 
that  no  growth  has  taken  place.  Ponfick,  Johne,  Rot- 
ter, Liming,  and  Hanan  claim  to  have  obtained  positive 
results  in  animals,  but  according  to  Bostrom  these  re- 
sults are  not  conclusive.  The  animals  used  for  experi- 
mentation have  been  calves,  swine,  dogs,  rabbits,  and 
guinea-pigs,  the  places  of  inoculation  being  the  anterior 
chamber  of  the  eye,  the  subcutaneous  intercellular  tissue, 
the  peritoneum,  and  the  blood;  and  the  material  em- 
ployed for  inoculation,  pus  from  the  infected  regions  in 
animals  and  man;  very  rarely  cultures. 

A  number  of  other  streptothrices  have  been  described 
in  connection  with  pathogenic  processes,  but  most  of 
them  are  not  well  defined.  They  have  been  found  in 
brain  abscess,  cerebro-spinal  meningitis,  pneumonic 
areas,  and  in  other  pathological  conditions.  Eppinger 
injected  cultures  into  guinea-pigs  and  rabbits,  and  ob- 
served that  it  caused  a  typical  pseudotuberculosis.  Con- 
solidation of  portions  of  both  lungs,  thickening  of  the 
peritoneum,  and  scattered  nodules  resembling  tubercles, 
were  noted  in  a  case  of  human  infection  as  due  to  a 
streptothrix  by  Flexner,  in  which  the  pathological 
picture  of  the  disease  resembled  so  nearly  tuberculosis 
in  human  beings  that  the  two  diseases  could  be  sepa- 
rated only  by  the  causative  micro  organism  in  each  case. 

THE  FUNGI. 

Most  of  the  fungi  are  not  pathogenic  and  interest  us 
merely  as  organisms  which  are  apt  to  infect  our  bac- 
teriological media.  Some  are,  however,  true  parasites, 
and  already  we  know  that  ringworm,  favus,  thrush,  and 
pityriasis  versicolor  are  caused  by  fungi.  Only  those 
causing  ringworm  and  favus  can  be  touched  on  here. 


620  APPENDIX. 

TRICHOPHYTON  (Ringworm  Fungus). 

Kingworm  of  the  body  or  hairless  parts  of  the  skin, 
tinea  circinata,  and  ringworm  of  the  hairy  parts,  tinea 
tonxurans  and  tinea  barbce  or  tinea  sycosis  are  due  to 
the  fungus  trichophyton,  discovered  by  Gruby  in  the 
human  hair,  and  between  the  epidermal  cells  by  Hebra, 
and  obtained  in  free  cultures  by  gravity. 


FIG.  83. 


Hair  riddled  with  ringworm  fungus.    Megalosporon  variety. 

According  to  Sabouraud,  whose  conclusions  are  based 
on  an  extensive  series  of  microscopical  examinations  of 
cases  of  tinea  in  man  and  animals,  of  cultivation  in 
artificial  media,  and  of  inoculation  on  man  and  animals, 
there  are  two  distinct  types  of  the  fungus  trichophyton 
causing  ringworm  in  man — one  with  small  spores  (2  to 
3  mm.),  which  he  calls  "T.  microsporon,"  and  one 
with  large  spores  (7  to  8  mm.),  which  he  calls  "T. 
megalosporon."  They  differ  in  their  mode  of  growth 
on  artificial  media  and  in  their  pathological  effects  on 


TRICHOPHYTON.  621 

the  human  skin  and  its  appendages.  T.  microsporon 
is  the  common  fungus  of  tinea  tonsurans  of  children, 
especially  of  those  cases  which  are  rebellious  to  treat- 
ment, and  its  special  seat  of  growth  is  in  the  substance 
of  the  hair.  T.  megalosporon  (Fig.  83)  is  essentially 
the  fungus  of  ringworm  of  the  beard  and  of  the  smooth 
parts  of  the  skin;  the  prognosis  as  regards  treatment  is 
good.  One-third  of  the  cases  of  T.  tonsurans  of  chil- 
dren are  due  to  trichophyton  megalosporon.  The  spores 
of  T.  microsporon  are  contained  in  a  mycelium;  but 
this  is  not  visible,  the  spores  appearing  irregularly 
piled  up  like  zoogloea  masses;  and,  growing  outside, 
they  form  a  dense  sheath  around  the  hair.  The  spores 
of  T.  megalosporon  are  always  contained  in  distinct 
mycelium  filaments,  which  may  either  be  resistant  when 
the  hair  is  broken  up,  or  fragile  and  easily  separating 
up  into  spores.  The  two  types  when  grown  in  artificial 
cultures  show  distinct  and  constant  characters.  The 
cultures  of  T.  microsporon  show  a  downy  surface  and 
white  color;  those  of  T.  megalosporon  a  powdery  sur- 
face, with  arborescent  peripheral  rays,  and  often  a  yel- 
lowish color.  Although  the  morphological  appearances, 
mode  of  growth,  and  clinical  effects  of  each  type  of  tri- 
chophyton show  certain  characters  in  general,  yet  there 
are  certain  constant  minor  differences  which  point  to 
the  fact  that  there  are  several  different  kinds  or  species 
of  fungus  included  under  each  type.  The  species  in- 
cluded under  T.  microsporon  are  few  in  number,  and, 
with  the  exception  of  one  which  causes  the  common 
contagious  " herpes"  of  the  horse,  almost  entirely 
human.  The  species  of  T.  megalosporon  are  numer- 
ous and  fall  under  several  natural  groups,  the  members 
of  which  resemble  one  another  both  from  clinical 


622 


APPENDIX. 


and  mycological  aspects  (Fig.  84).  Many  animals  are 
subject  to  the  growth  upon  their  skins  of  particular 
species  of  T.  megalosporon. 


FIG.  84. 


These  two  half-plates  show  three- mouths'  growth  on  peptone  maltose  agar  of 
two  megalosporon  varieties  of  the  ringworm  fungus.    Natural  size. 


ACHORION  SCHGENLEINII  (Favus). 

Favus  is  due  to  a  fungus  discovered  by  Schoenlein  in 

1839,  and  called  by  Remak  Achorion  schcenleinii.     The 

disease  is  communicated  by  contagion,  the  fungus  being 

often  derived  from  animals,  especially  cats,  mice,  rab- 


A  CHORWN  SCHCENLEINIL  623 

hits,  fowls;  and  dogs  are  also  subject  to  it.  It  grows 
much  more  slowly  than  the  ringworm  fungus,  and  is, 
therefore,  not  so  easily  transmitted.  Want  of  cleanli- 
ness is  a  predisposing  factor.  The  fungus  seems  to 
find  a  more  favorable  soil  for  its  development  on  the 
skin  of  persons  in  weak  health,  especially  from  phthisis, 
than  in  others. 

Pathologically,  the  disease  represents  the  reaction  of 
the  tissues  to  the  irritation  caused  by  the  growth  of  the 
fungus.  The  spores  generally  find  their  way  into  the 

FIG.  85. 


A  portion  ot  a  favus  infected  hair.    Magnified. 

hair-follicles,  where  they  grow  round  the  hair-seat 
(Fig.  85).  The  favus  fungus  grows  in  the  epidermis, 
the  density  of  the  growth  causing  pressure  on  the  parts 
below,  thus  crushing  out  the  vitality  of  the  hair  and 
giving  rise  to  atrophic  scarring.  The  disease  shows  a 
marked  preference  for  the  scalp,  but  no  part  of  the 
skin  is  exempt,  and  even  the  muco-us  membranes  are 
liable  to  be  attacked.  Kaposi  has  reported  a  case  in 
which  a  patient  suffering  from  universal  favus  died, 
with  symptoms  of  severe  gastro  intestinal  irritation, 
which  was  found  after  death  to  be  due  to  the  presence 


624  APPENDIX. 

of  the  favus  fungi  in  the  stomach  and  intestine.  On 
the  scalp  it  first  appears  as  a  tiny  sulphur-yellow 
disk  or  scutulum,  depressed  in  the  centre  like  a  cup  and 
pierced  by  a  hair.  This  is  the  characteristic  lesion.  The 
cup-shape  is  attributed  by  Unna  to  growth  at  the  sides 
proceeding  more  vigorously  than  at  the  centre. 

There  is  some  difference  of  opinion  as  to  whether 
there  is  only  one  or  several  varieties  of  favus  fungus. 
It  was  suggested  by  Quincke  that  there  are  three  dif- 
ferent species  of  favus  fungus.  Later  investigations 

FIG.  86. 


Five-months'  old  colony  of  favus  on  peptone  maltose  agar.    Actual  size. 

have  apparently  shown,  however,    that  the  achorion 
Schoenleinii  is  the  only  fungus  of  favus. 

The  favus  fungus  is  readily  cultivated  at  the  body- 
temperature,  and  also  at  room-temperature,  in  the  or- 
dinary culture  media,  as  agar,  blood-serum,  gelatin, 
bouillon,  milk,  infusion  of  malt,  eggs,  potato,  etc. 
(Fig.  86).  The  growth  develops  slowly  and  shows  a 
preference  to  grow  beneath  the  surface  of  the  medium — 
except  on  potato,  upon  which  it  develops  on  the  surface 
in  layers.  The  characteristic  form  of  growth  is  that  of 
moss-like  projections  from  a  central  body.  The  color 


YEASTS.  625 

is  at  first  grayish-white,  then  yellowish.  As  seen 
under  the  microscope,  ray-like  mycelium  filaments  are 
developed,  which  divide  into  branches.  The  ends  are 
often  swollen  or  club-shaped,  and  there  are  various 
enlargements  along  the  body  of  the  filament. 

YEASTS  (Saccharomyces). 

These  micro-organisms  are  of  the  greatest  importance 
in  brewing  and  baking,  but  as  yet  no  important  patho- 
logical lesions  in  man  have  been  attributed  to  them, 
although  certain  recent  experiments  have  shown  that 
some  varieties  when  injected  are  capable  of  producing 
tumors  and  many  are  pathogenic  for  mice.  They  are 
are  not  uncommonly  present  in  the  air  and  in  cultures 
made  from  the  throat.  They  consist  of  round  or  oval 
cells,  usually  many  times  larger  than  the  bacteria. 
They  usually  reproduce  themselves  by  budding,  a  por- 
tion of  the  protoplasm  budding,  and  finally  being  cut 
off  to  form  a  new  individual. 


40 


CHAPTER  XXXVIII. 

PLASMODIUM  MALARI^E  (MALARIAL  PARASITES  ; 
LAVERANIA) — AMCEBA  COLI  (AMCEBA  DYSEN- 
TERIC OF  COUNCILMAN  AND  LAFLEUR  ;  DYSEN- 
TERIC AMCEBA). 

MANY  attempts  have  been  made  from  time  to  time 
to  discover  a  specific  organism  in  malaria.  As  early  as 
1846,  according  to  Marchiafava  and  Bignami,  an  Ital- 
ian observer  (Risori)  suggested  the  possible  parasitic 
nature  of  the  disease.  In  1880,  Laveran  announced  the 
discovery  of  certain  parasitic  bodies  in  the  blood  of 
patients  with  malarial  fever.  He  recognized  that  they 
were  parasites,  and  raised  the  question  whether  they 
were  amoebae.  Subsequently,  influenced  no  doubt  by 
the  presence  of  the  motile  filaments,  he  suggested  the 
term  osdllaria  malarice.  Marchiafava  and  Celli  de- 
scribed with  great  accuracy  the  intracorpuscular  amoe- 
boid form,  to  which  they  gave  the  name  plasmodium. 
The  most  important  additional  fact  was  added  by  Golgi, 
who  pointed  out  the  association  of  the  paroxysm  with 
the  segmentation  of  a  group  of  the  malarial  organisms. 
Laveran' s  work  and  the  differentiation  by  the  Italian 
observers  of  varieties  of  the  parasite  in  different  clinical 
forms  of  the  disease  have  since  received  full  confirma- 
tion, and  the  testimony  is  now  unanimous  in  France, 
England,  India,  America,  Italy,  and  Germany  that 
these  bodies  are  always  present  in  the  malarial  fevers. 


PLASMODIUM  MALARIA.  627 

There  is  still  much  uncertainty  with  regard  to  the 
classification  of  the  parasites.  Many  authors  place  them 
among  the  sporozoa  in  the  order  of  the  hoemosporidia  of 
Danilewsky;  others  place  them  in  the  aarcodinia,  and 
speak  of  them  as  hcemamcebce.  Until  the  matter  is  settled, 
however,  they  may  be  considered  to  belong  to  the  gen- 
eral order  of  protozoa  and  to  that  group  of  organisms 
known  as  hcematozoa.  Parasites  of  the  red  blood-cor- 
puscles have  been  met  with  abundantly  in  the  blood  of 
fish,  turtles,  and  many  species  of  birds. 

The  relation  of  the  different  forms  of  the  malarial 
parasite  to  each  other  and  to  the  varieties  of  the  disease 
are  still  under  discussion.  Galgi,  Marchiafava  and 
other  Italian  observers  hold  that  they  are  distinct 
varieties,  not  interchangeable,  though  closely  allied 
biologically.  Laveran,  on  the  other  hand,  contends  for 
the  unity  of  the  forms,  which  he  regards  as  modifica- 
tions of  one  polymorphic  parasite.  But  with  the 
present  imperfect  knowledge  of  the  full  life-history  of 
the  parasite  the  question  cannot  be  considered  as  settled. 

The  following  varieties  are  associated  with  the  differ- 
ent forms  of  malarial  fever  : 

I.  Parasite  of  the  Simple  Intermittent  Fever,  (a)  TER- 
TIAN PARASITE  (see  Plate  II.).  If  the  blood  of  a 
patient  be  examined  during  or  shortly  after  the  chill 
in  tertian  fever,  inside  the  red  blood-corpuscles,  or  less 
often  free  in  the  plasma,  will  be  seen  small,  pale,  hya- 
line amoebae  which  undergo  rapid  changes  in  shape, 
often  assuming  the  form  of  a  star  or  of  a  cross.  There 
may  be  no  pigment  visible,  and  to  these  hyaline  bodies 
Marchiafava  and  Celli  gave  the  name plasmodia.  In  a 
few  examples  scattered  pigment  granules  may  be  seen 
in  the  amcebse,  usually  placed  near  the  periphery.  In 


628  APPENDIX. 

dry  specimens  these  bodies  stain  deeply  with  methylene- 
blue,  and  they  are  solid  or  vesicular  in  form.  If  the 
examination  be  made  within  twelve  to  eighteen  hours 
after  the  chill  the  hyaline  bodies  are  seen  to  have 
grown  to  occupy  one-fourth  to  one-third  of  the  bodies 
of  the  red  cells.  They  are  more  pigmented,  and  the 
corpuscles  containing  them  have  become  gradually  paler 
and  somewhat  expanded.  The  pigment  granules,  which 
at  first  are  small,  increase  in  size,  and  the  organisms 
show  very  active  amoeboid  movements.  At  the  end  of 
forty-eight  hours  they  occupy  entire  corpuscles,  are  very 
sluggish  in  their  movements,  and  look  like  thin,  trans- 
lucent shells,  and  are  usually  devoid  of  color.  Many 
of  the  organisms  then  undergo  the  remarkable  change 
known  as  segmentation,  which  precedes  and  is  asso- 
ciated with  chills  and  fever.  The  amoeboid  movement 
ceases  as  well  as  that  of  the  pigment  granules.  The 
latter  gradually  collect  toward  the  centres  of  the  amoebae 
until  they  are  in  the  form  of  closely  packed,  more  or  less 
central  clumps.  The  protoplasm  becomes  more  finely 
granular,  and  indistinct  lines  of  striation  are  seen,  which 
begin  at  the  periphery.  At  this  stage  the  organisms 
may  present  the  appearance  of  rosettes.  The  segmen- 
tation progresses  until  the  entire  protoplasm  is  divided 
into  twelve  to  eighteen  or  twenty  spheres.  The  shell 
of  the  corpuscles  containing  a  parasite  usually  bursts, 
and  the  small,  rounded,  hyaline  bodies  are  set  free. 
Each  one  of  these  little  bodies  consists  of  a  translucent 
protoplasm,  with  a  central,  more  highly  refractile  spot. 
In  stained  preparations,  during  the  segmenting  process, 
the  reticulum  becomes  denser  and  sharper,  and  then 
breaks  up  into  fifteen  to  twenty  small  spheroidal 
spores. 


PLASMODIUM  MALARIJE.  *  629 

The  segmentation  is  regarded  as  a  reproductive  pro- 
cess, and  these  small  spherical  bodies  are  believed  to 
be  the  spores  which  penetrate  a  new  set  of  corpuscles, 
and  so  begin  a  new  cycle  of  development.  The  pig- 
ment is  discharged  into  the  plasma,  and  partly  taken 
up  by  the  leucocytes.  It  is  finally  lodged  chiefly  in 
the  spleen,  liver,  and  lymphatic  organs.  The  pres- 
ence of  the  segmenting  forms  is  invariably  associated 
with  the  paroxysm.  On  finding  them  in  the  blood  it 
can  be  predicted  with  certainty  that  a  paroxysm  is 
imminent.  In  quotidian  fever  we  have  to  deal  with 
two  groups  of  tertian  (or  three  groups  of  quartan)  para- 
sites, maturing  on  successive  days;  and  the  full-grown 
segmenting  forms  of  to-day's  paroxysms  and  the  half- 
grown  organisms  of  to-morrow's  attack  are  to  be  found 
in  the  blood. 

(b)  QUARTAN  PARASITE  (see  Plate  II.).  The  early 
forms  within  the  red  blood-corpuscles  are  amoeboid 
bodies,  similar  to  those  of  tertian  fever.  Soon,  how- 
ever, it  is  noticed  that  the  pigment  is  different ;  the 
granules  are  larger  and  blacker,  and  the  amoeboid 
movements  are  not  so  active.  In  their  growth  the 
parasites  do  not  decolorize  the  corpuscles,  which  some- 
times have  a  greenish,  brassy  look.  From  the  sixty- 
fourth  to  the  seventy-second  hour  the  amoebae  have 
reached  their  full  development,  occupying  the  greater 
portion  of  the  affected  corpuscles  ;  but  a  thin  rim  of 
colored  stroma  can  usually  be  seen.  Some  of  the  cor- 
puscles are  completely  filled  by  the  parasites.  The 
cells,  as  a  rule,  appear  shrunken  rather  than  swollen. 
Even  at  this  stage  a  skilled  observer  can  usually 
recognize  the  quartan  from  the  tertian  organism.  The 
pigment  granules  then  collect  toward  the  centre,  and 


630  APPENDIX. 

in  so  doing  usually  form  distinct  rays.  Then,  as  in 
the  tertian  form,  the  organism  begins  to  segment ;  a 
marginal  indentation  is  first  seen,  with  lines  of  radia- 
tion, and  a  beautiful  rosette  is  formed,  which  segments 
into  from  six  to  ten,  occasionally  twelve,  small,  spher- 
ical or  ovoid  bodies.  The  character  of  the  pigment, 
the  smaller  size  of  the  organism,  and  the  development 
are  differences  which  separate  the  quartan  from  the 
tertian  variety. 

In  the  quartan  malarial  fever  the  blood  may  show 
two  or  more  groups  of  parasites.  There  may  be  two 
groups  which  reach  maturity  on  successive  days,  with 
one  day  interval — double  quartan  fever;  or  there  may 
be  three  groups  of  organisms  maturing  on  successive 
days,  causing  daily  paroxysms — triple  quartan  fever. 

II.  The  -ffistivo-autumnal  Parasite  (see  Plate  II.). 
In  the  more  irregular  and  as  a  rule  pernicious  types 
of  malarial  infection  which  are  met  with  in  the  autumn 
months  a  third  variety  of  organism  may  be  recognized, 
which  has  been  specially  studied  by  the  Italian  obser- 
vers. The  youngest  forms  of  this  parasite  are  small 
hyaline  bodies  about  one-sixth  the  diameter  of  the  red 
cell.  At  first  they  are  quiescent,  but  later  develop 
active  amoeboid  movement.  They  are  at  this  stage 
not  unlike  those  of  the  tertian  varieties;  but  the  hya- 
line body  is  more  signet  ring-like,  more  highly  refrac- 
tile,  and  the  central  part  often  looks  shaded,  as  if  a 
more  solid  body  were  enclosed  within  a  vacuole.  As 
this  form  increases  the  amoeboid  movements  are  well 
seen.  The  pigment  is  in  small  amount,  at  first  in  the 
form  of  one  or  two  very  dark  granules  at  the  margin 
of  the  amoebae,  and  the  pigment  never  becomes  so 
abundant  as  in  the  tertian  or  quartan  forms.  The 


PLASMODIUM  MALARIJE.  631 

organism  rarely  occupies  more  than  about  one-third  of 
the  corpuscle,  the  stroma  of  which  is  never  entirely  de- 
colorized. On  the  contrary,  it  often  presents  a  curious 
brassy-green  appearance,  and  looks  shrunken  or  crum- 
pled. The  cycle  of  development  of  this  form  is  rarely 
carried  out  entirely  in  the  circulating  blood,  but  the 
bodies  with  centrally  placed  pigment  are  not  uncom- 
mon. The  observations  of  the  Italian  observers  seem 
to  show  conclusively  that  the  segmentation  takes  place 
in  the  spleen  and  in  the  bone-marrow  and  internal 
organs.  The  length  of  its  cycle  of  development  has  not 
been  determined.  Probably  different  groups  mature  at 
varying  intervals  of  time,  from  twenty-four  hours  or 
less  to  forty-eight  or  more  (Welch).  The  fever  associ- 
ated with  this  organism  is  characterized  by  irregularity, 
the  paroxysms  are  not  at  definite  periods,  and  the 
pyrexia  may  be  more  or  less  continuous,  with  remissions. 
This  form  is  associated  with  the  severer  types  of  the 
malaria  seen  in  late  summer  and  autumn — the  sestivo- 
autumnal  fevers  of  Cuba,  Italy,  etc. 

There  are  several  other  points  of  interest  about  the 
parasites.  A  corpuscle  containing  a  half -grown  organism 
may  suddenly  rupture;  the  haemoglobin  diffuses,  and 
the  pigmented  parasite  is  set  free.  The  parasite  may 
break  up  into  two  or  three  portions,  perhaps  from 
pressure  on  the  slide,  and  slight  amoeboid  changes  may 
be  seen.  In  other  instances,  apparently  from  certain 
free  extra-corpuscular  organisms,  the  remarkable  flagel- 
late form  develops  itself.  The  pigment  becomes  more 
central,  and  the  granules  dance  with  great  activity. 
Suddenly,  long,  thread-like  processes  extend  from  the 
body  of  the  parasite  and  display  remarkable  move- 
ments, thrashing  about  over  the  corpuscle  with  extra- 


632  APPENDIX. 

ordinary  rapidity.  A  flagellum  may  break  off  from 
the  main  body  and  move  about  independently  among 
the  corpuscles.  While  these  flagellate  bodies  appear 
in  both  the  tertian  and  quartan  fevers  they  are  very 
much  more  numerous  in  the  irregular  malaria.  The 
significance  of  the  flagellate  form  is  still  under  discus- 
sion. By  some  it  has  been  regarded  as  a  degenerate 
form. 

In  the  sestivo-autumnal,  quotidian,  or  pernicious  mala- 
rial fevers  there  is  developed  also  a  very  striking  body, 
to  which  much  attention  has  been  paid,  viz.,  the  <e  cres- 
cent "  of  Laveran.  In  any  case  of  irregular  malarial 
fever  which  has  lasted  a  week  or  more  these  bodies  are 
to  be  found.  They  are  developed  within  the  red  blood- 
corpuscle,  the  margin  of  which  may  usually  be  seen  on 
the  concave  surface  of  the  crescent.  The  border  is  very 
sharply  defined,  the  protoplasm  uniform,  homogenous, 
with  coarse  pigment  granules,  often  in  the  form  of  rods, 
which  are  collected  about  the  centre.  Bodies  similar 
in  structure,  but  differing  in  form,  being  ovoid  and 
rounded,  are  also  met  with ;  and  the  change  of  a  cres- 
cent into  an  ovoid  or  rounded  body  can  be  traced,  which, 
in  turn,  may  in  some  instances  be  seen  to  project  flagella 
or  form  a  flagellated  body  similar  to  that  derived  from 
the  extracorpuscular  organisms  above  referred  to. 
Most  authors  say  that  both  kinds  of  flagellate  bodies 
do  not  develop  unless  the  blood  be  exposed  to  the  air, 
but  an  exposure  of  one  or  two  minutes  gives  the  best 
results.  It  would  seem  that  they  do  not  exist  as  flagel- 
late forms  in  the  circulation.  (Osier,  in  Allbutt's  Sys- 
tem of  Medicine.) 

Pigmented  Leucocytes.  Typical  pigmented  leucocytes 
are  very  characteristic  signs  in  malarial  blood,  and  on 


PLASMODIUM  M ALARMS.  633 

their  presence  alone  the  diagnosis  must  often  rest:  (1) 
In  severe  acute  cases  after  the  administration  of  much 
quinine;  (2)  in  remittent  malarial  fevers;  and  (3)  in 
chronic  malarial  fever  and  cachexia.  They  persist  in 
the  blood  long  after  all  traces  of  parasites  have  dis- 
appeared. The  identification  of  free  malarial  pigment 
is  usually  hazardous,  and  the  diagnosis  of  malaria 
should  never  be  based  on  its  presence  alone  (Ewing). 

Inoculation  Experiments.  Malarial  infection  can  be 
transmitted  directly  from  man  to  man  by  subcutaneous 
or  intravenous  inoculation  of  malarial  blood.  This  was 
shown  first  by  Gerhardt  in  1884.  Later  experiments, 
chiefly  by  Italians  observers,  have  confirmed  Gerhardt7  s 
investigations,  and  almost  in  every  instance  the  variety 
of  organism  introduced  has  been  reproduced.  It  has 
also  been  experimentally  shown  that  the  ague  paroxysm 
is  associated  with  the  segmentation  of  enormous  groups 
of  intracorpuscular  amoeba?,  the  symptoms  being  prob- 
ably due,  as  Bacelli  suggests,  to  toxins  liberated  during 
sporulation  or  to  substances  set  free  in  the  blood  by 
the  rapid  destruction  of  a  large  number  of  its  corpuscles. 
The  period  of  incubation  is  from  eleven  to  twelve  days 
in  the  regular  intermittents  and  from  two  to  five  days 
in  the  irregular  autumnal  fever. 

Active  phagocytosis  goes  on  in  all  forms  of  malarial 
infection,  but  its  true  significance  is  still  undetermined. 
That  many  parasites  are  devoured  by  the  leucocytes, 
especially  in  the  spleen,  is  certain.  This  apparently 
takes  place  during  or  after  sporulation.  But  sponta- 
neous recovery  may  also  be  due  to  the  death  of  the 
plasmodia.  It  is  not  improbable,  however,  that  the 
phagocytes  contribute  to  the  process  of  recovery,  even 
if  they  are  not  the  chief  factors  in  it. 


634  APPENDIX. 

With  regard  to  immunity,  we  know  that  one  attack 
of  malaria  may  linger  a  long  time,  and  seems  rather  to 
favor  than  to  prevent  a  new  infection.  There  is, 
however,  a  natural  susceptibility  to  the  disease  which 
is  very  variable.  Different  races  of  men  especially 
seem  to  possess  in  variable  degree  the  power  of  resist- 
ance to  malarial  infection.  This  is  shown  not  only  in 
a  diminished  tendency  to  contract  the  disease,  but  also 
in  the  form  by  which  they  are  affected.  For  instance, 
the  negroes  in  the  Southern  parts  of  the  United  States 
are  much  less  liable  to  contract  malaria  than  the  whites; 
and  Martin  reports  that  the  Europeans  living  in  Suma- 
tra are  far  more  frequently  and  severely  affected  by 
malaria  than  the  natives,  who,  if  they  are  attacked  at 
all,  it  is  only  with  the  simple  intermittent  tertian  and 
quartan  fevers. 

The  Action  of  Quinine  on  the  Parasites.  Laveran 
showed  that  a  solution  of  1  to  10,000  of  quinine,  run 
under  the  cover-glass,  would  check  at  once  the  move- 
ments of  malarial  organisms.  As  demonstrated  by 
Marchiafava  and  Celli,  however,  a  like  effect  is  pro- 
duced either  by  the  water  or  by  the  salt  solution  in 
which  the  quinine  is  dissolved,  and  we  meet  with  an 
almost  insuperable  difficulty  in  the  study  of  the  direct 
action  of  the  drug  upon  the  parasites  themselves. 

Many  careful  experiments  have  been  made  to  deter- 
mine the  effect  of  quinine  on  the  parasites  circulating 
in  the  blood,  and  Romanowsky,  Golgi  and  others  have 
reported  a  diminution  in  the  activity  of  the  amoeboid 
movements.  Osier  stated  that,  as  a  result  of  careful 
hourly  examinations  made  in  a  series  of  cases  with  a 
view  of  ascertaining  the  direct  influence  of  full  doses  of 
quinine,  he  was  unable  to  make  up  his  mind  that  any 


PLASMODIUM  MALARIA.  635 

particular  change  took  place  in  the  intracorpuscular 
tertian  parasite  while  undergoing  destruction  by  the 
specific. 

The  following  points,  nevertheless,  about  the  action 
of  quinine  on  the  parasites  seem  to  be  well  established  : 
First,  that  under  its  use  the  intracorpuscular  varieties, 
whether  tertian,  quartan,  or  sestivo-autumnal,  rapidly 
disappear  from  the  circulating  blood;  second,  that 
quinine  administered  some  hours  before  a  paroxysm 
will  not  interrupt  the  cycle  of  their  development,  but 
will  usually  destroy  the  products  of  segmentation,  and 
so  check  the  succeeding  paroxysm;  third,  that  the  cres- 
centic  and  ovoid  bodies  which  develop  in  sestivo- 
autumnal  fevers  are  very  slightly  affected  by  the  action 
of  quinine. 

Mixed  Infection  in  Malarial  Fever.  It  is  now  a  well- 
known  fact  that  along  with  a  malarial  infection  there 
may  exist  another  due  to  the  typhoid  bacillus,  to  one 
of  the  pyogenic  cocci,  or  to  other  micro-organisms. 
Such  mixed  infection  may  make  a  complete  diagnosis 
a  very  difficult  matter. 

Diagnosis.  The  diagnosis  of  malaria  in  all  its  forms 
has  been  greatly  simplified  by  Laveran's  discovery. 
This  is  not  a  matter  of  so  much  importance  in  the 
simple  typical  intermittents,  but  in  the  atypical  forms 
of  the  disease,  and  especially  in  pernicious  malaria,  the 
symptoms  of  which  are  readily  overlooked,  serious 
errors  in  diagnosis  may  be  made.  Moreover,  par- 
oxysms of  intermitting  fever,  which  are  common  in 
other  diseases,  may  be  mistaken  for  those  of  malaria 
— such  as  occur  in  the  early  stages  of  tuberculosis,  in 
ulcerative  endocarditis,  in  suppuration  associated  with 
septicaemia  or  pyaemia,  in  pyelitis;  etc.  In  all  such 


636  APPENDIX. 

cases,  and  in  cases  of  mixed  malarial  infection  occur- 
ring in  malarial  regions,  a  careful  blood  examination 
enables  a  positive  diagnosis  to  be  made  in  a  large 
majority. 

Technique  of  Blood  Examinations  for  Malaria.  The 
finding  of  the  parasite  should  not  prevent  us  from 
seeking  farther  in  doubtful  cases  by  means  of  the 
Widal  reaction  and  blood  cultures  for  other  infections 
which  may  exist  along  with  the  malaria. 

The  parasites  require  a  proper  technique  and  a 
certain  experience  for  their  recognition.  The  fresh 
blood,  when  it  can  be  obtained,  should  be  examined, 
but  if  no  bodies  be  found,  stained  preparations  should 
always  be  later  searched  through;  the  drops  may  be 
taken  either  from  the  tip  of  the  finger  or  from  the 
lobe  of  the  ear.  It  is  important  to  have  a  perfectly 
clean  cover-glass  and  slide,  and  to  cleanse  the  skin 
thoroughly  and  to  wipe  it  dry,  so  as  to  avoid  dirt  and 
perspiration.  A  very  small  drop  should  be  taken,  and 
care  must  be  exercised  that  the  cover-slips,  when  pressed 
against  the  blood-drop,  do  not  touch  the  skin.  The 
drop  should  be  so  small  that  the  corpuscles  are  spread 
out  in  a  uniform  layer  and  are  not  in  rolls  when  the 
cover-glass  is  laid  upon  the  slide,  for  the  intracorpus- 
cular  form  cannot  be  well  seen  unless  the  blood-disk 
presents  the  flattened  surface.  For  making  permanent 
preparations  the  blood  is  collected  upon  cover-glasses 
in  very  thin  films,  which  should  be  instantly  dried. 
The  blood-cells  are  fixed  by  immersion  in  equal  parts 
of  alcohol  (95  per  cent.)  and  ether  for  fifteen  minutes, 
or  by  exposing  for  five  minutes  over  a  wide-mouthed 
bottle  containing  25  per  cent,  solution  of  formalin,  or 
by  heating  to  120°  C.  for  ten  minutes.  Ewing  advises 


PLASMODIUM  MALARIA.  637 

the  alcohol  and  ether  method.  The  preparations  are 
stained  with  methylene-blue,  or,  if  desired,  with  a 
double  stain  of  methylene-blue  and  eosin.  The  prep- 
aration is  covered  with  a  mixture  made  of  equal  parts 
of  a  saturated  alcoholic  solution  of  eosin  and  water  for 
one  minute  ;  wash  in  water  and  dry  in  air.  The  prep- 
aration is  then  covered  by  a  saturated  watery  sol  ution  of 
methylene-blue  for  a  minute  or  two,  washed  in  water, 
dried,  mounted,  and  examined  with  the  immersion  lens. 
Thorough  drying  after  the  eosin  staining  makes  the 
blue  stain  of  the  parasites  sharper  (Ewing). 

In  some  cases  of  sestivo-autumnal  fever  the  para- 
sites are  chiefly  in  the  spleen,  liver,  and  bone-marrow. 
The  blood  withdrawn  directly  from  the  spleen  may 
show  large  numbers,  although  in  the  circulating  blood 
they  may  be  scanty.  In  these  cases  puncture  of  the 
spleen  and  examination  of  the  blood  withdrawn  may 
render  the  diagnosis  more  certain,  but  in  acute  splenic 
tumor  the  procedure  is  not  without  risk.  The  finding 
of  malarial  parasites  in  the  blood  not  only  separates  the 
intermittent,  continued,  and  remittent  malarial  fevers 
from  all  other  diseases  in  which  similar  fevers  may 
occur,  but  the  variety  of  parasites  found  influences  the 
prognosis  of  the  malarial  infection.  The  number  of 
parasites  observed  on  examination  also  influences  the 
prognosis  to  a  certain  degree,  though  too  great  weight 
should  not  be  laid  on  this  point,  particularly  as  the 
result  of  a  single  examination.  Whether  there  are  any 
forms  of  malarial  infection  in  which  there  are  no  plas- 
modia  present  in  the  circulating  blood  is  a  question 
for  future  determination.  We  know  that  in  all  severe 
seizures,  if  the  blood  is  examined  within  twenty-four 
hours  of  the  beginning  of  the  paroxysms  and  before 


638  APPENDIX. 

much  quinine  is  given,  the  plasm odia  can  readily  be 
found,  usually  in  considerable  numbers.  In  some  very 
mild  initial  paroxysms  the  plasm  odia  may  be  difficult 
to  find.  In  aestivo-auturnnal  malaria,  while  quinine  is 
being  administered,  there  may  be  no  organisms  during 
the  period  between  the  second  and  fourth  day,  but  on 
the  fourth  or  fifth  day  the  crescents  almost  always  make 
their  appearance,  notwithstanding  the  use  of  quinine 
(Ewing). 

Mode  of  Infection.  It  is  generally  acknowledged 
that  the  most  common  mode  of  infection  in  malaria 
is  through  the  air.  Whether  the  disease  may  be 
directly  conveyed  by  water  has  been  much  disputed. 
Many  favor  the  view,  but  experimental  evidence  is 
distinctly  against  it.  Persons  have  been  allowed  to 
drink  water  from  the  Pontine  marshes  without  ill 
effects,  and  in  Bacelli's  clinic  at  Rome  experiments 
were  made  in  thirty  cases  with  water  from  malarial 
districts  without  a  single  positive  result.  Grassi  could 
not  produce  the  disease  with  dew  from  malarial  regions 
or  by  allowing  healthy  men  to  drink  blood  from  mala- 
rial patients.  We  may  therefore  assume  that  malarial 
infection  is  not  produced,  as  a  rule,  by  way  of  the  in- 
testines. Numerous  experiments  have  shown,  oir  the 
contrary,  that  the  infection  may  be  induced  by  subcu- 
taneous inoculation.  It  is  quite  conceivable,  therefore, 
that  under  natural  conditions  malarial  infection  may  be 
produced  by  way  of  the  skin,  and  possibly  by  the  bites 
of  insects.  This  is  all  the  more  probable,  as  certain 
varieties  of  mosquitoes,  in  malarial  regions,  have  been 
found  to  be  laden  with  the  plasmodia.  In  another  wide- 
spread disease  produced  by  blood  parasites — Texas  fever 
in  cattle — it  has  been  shown  that  the  amoebae  are  con- 


PLASMODIUM  MALARIJE.  639 

veyed  by  means  of  the  cattle  tick  from  animal  to  animal. 
The  further  the  investigations  have  been  pushed  the 
closer  becomes  the  connection  between  mosquitoes  and 
malarial  infection  in  man.  So  far  as  we  know,  a  few 
varieties  of  mosquitoes  and  man  are  the  only  places 
where  the  malarial  parasites  develop,  and  Koch,  fol- 
lowing lines  suggested  by  the  work  of  others,  has 
now  shown  that  the  fresh  cases  of  infection  with 
malaria  occur  only  in  warm  weather  when  the  parasites 
can  develop  in  the  mosquitoes.  Koch's  idea  is  that 
human  beings  having  chronic  malaria  preserve  in  their 
blood  the  malarial  parasites  during  the  cool  months. 
In  the  warm  weather  mosquitoes  become  infected,  the 
parasites  develop  in  them  and  are  present  in  their 
poison  sacs.  These  mosquitoes  bite  and  infect  fresh 
human  cases  through  subcutaneous  inoculation.  He 
believes  if  we  would  treat  all  chronic  malarial  patients 
with  quinine  so  as  to  prevent  the  development  of  the 
parasites  and  thus  the  infection  of  the  new  crop  of 
mosquitoes,  we  would  prevent  most,  at  least,  of  human 
infection. 

Blood  parasites  are  extremely  common  in  cold- 
blooded animals,  fish,  reptiles,  and  in  birds.  .Birds 
appear  to  suffer  from  malarial  infection  similar  to  that 
in  man,  and  the  parasites  found  in  the  blood-corpuscles 
are  closely  allied  to  those  of  human  malaria.  But  in 
birds  infection  cannot  be  produced  by  subcutaneous  or 
intravenous  inoculation  with  parasites  from  human 
blood,  nor  can  infection  be  transmitted  from  birds  to 
man.  The  blood  parasites  found  in  fish  and  reptiles, 
though  similar  to,  are  not  identical  with,  those  found 
in  man,  and  they  are  not  transmissible  to  man.  This 
source  of  infection  may,  therefore,  be  excluded.  Ex- 


640  APPENDIX. 

perience  shows  that  the  disease  is  Dot  contagious,  in 
the  ordinary  sense  of  the  word,  and  that  it  is  not 
directly  transmitted  from  man  to  man. 

AMCEBA   COLI  (Amoeba  Dysenteriae  of  Councilman  and 
Lafleur;  Dysenteric  Amoeba). 

In  1875,  Losch,  of  St.  Petersburg,  gave  the  first 
accurate  description  of  an  amoeboid  organism  which 
he  found  in  the  stools  of  a  dysenteric  patient,  and  to 
it  he  gave  the  name  amoeba  coli.  He  claimed  that  this 
organism  is  the  cause  of  dysentery,  and  he  succeeded  in 
producing  a  superficial  ulceration  of  the  large  intestine 
in  one  of  four  dogs  which  had  received  rectal  injec- 
tions of  the  dysenteric  stools.  Losch' s  observation 
has  been  confirmed  by  various  researches  in  different 
countries. 

Morphology.  The  amoeba  is  a  unicellular  organism 
belonging  to  the  class  of  rhizopada  of  the  protozoa, 
and  consists  of  slightly  differential  masses  of  proto- 
plasm, which,  under  favorable  circumstances,  exhibits 
spontaneous  movements.  In  a  state  of  rest  the  amoeba 
assumes  a  spherical  shape  which  appears  discoid  in  the 
field  of  the  microscope.  It  may  generally  be  distin- 
guished from  the  other  cellular  elements  found  in  the 
feces  by  its  pale  greenish  tint  and  by  its  stronger 
refraction  of  light.  Its  diameter  varies  within  wide 
limits,  6//  to  35//7  more  commonly  between  12//  and 
26//.  It  is  noteworthy  that  such  differences  in  size 
are  found,  as  a  rule,  in  different  cases  of  the  disease, 
while  the  amoebae  in  any  individual  case  are  nearly 
uniform  in  diameter.  The  body  of  a  resting  amoeba 
has  a  well-defined,  regular  body,  which,  under  ordi- 
nary conditions,  appears  as  a  thin,  single,  dark  line. 


PLATE   II. 


v 


o 


Figs,  i,  2,  and  3  show  three  phases  of  the  parasite  of  tertian  fever. 
Fig.  i,  ring  form,  showing  beginning  pigment  formation.  Fig.  2,  full- 
grown  parasite.  Fig.  3,  segmenting  bodies.  (WELCH  and  THAYER.) 

Figs.  4,  5,  and  6  show  the  parasite  of  quartan  fever  at  different  stages 
of  growth.  Fig.  4,  moderately  developed  intracorpuscular  parasite.  Fig.  5, 
large  swollen  extracorpuscular  form.  Fig.  6,  flagellate  body. 

(WELCH  and  THAYER.) 

Figs.  7,  8,  and  9  illustrate  the  sestivo-autumnal  parasite.  Fig.  7,  ring-like 
body,  with  a  few  pigmented  granules.  Fig.  8,  crescent  still  in  blood-cor- 
puscle. Fig.  9,  vacuolation  of  crescent.  (WELCH  and  THAYER.) 

Fig.  10.  Amoeba  from  section  of  intestine  hardened  in  alcohol  and 
stained  with  methylene  blue.  (COUNCILMAN  and  L,AFLEUR.) 


AMCEBA  COLL  641 

The  body  consists  of  two  portions:  the  inner  one, 
which  is  more  or  less  granular  and  of  a  darker  color, 
is  known  as  the  entoplasma;  the  outer  one,  which  is 
homogeneous  and  of  a  lighter  color,  as  the  ectoplasma 
(see  Plate  II. ,  Fig.  10).  This  division  into  two  zones 
cannot  always  be  made  out,  and  is  more  evident  in  the 
motile  than  in  the  resting  amoeba. 

The  entoplasma  constitutes  the  greater  portion  of  the 
body  of  the  amoeba,  being  usually  centrally  situated, 
but  occasionally  slightly  eccentric.  In  the  smaller 
forms  of  amoebae  it  is  finely  granular,  and  may  show 
no  other  structure.  In  the  larger  forms  it  is  more 
coarsely  granular,  and  often  contains  clear,  circular, 
and  slightly  oval  spaces  known  as  vacuoles.  These 
are  extremely  variable  in  number  and  size. 

The  edoplasma  is  quite  homogeneous,  forming  a  zone 
of  variable  thickness  around  the  entoplasrn.  It  has 
the  appearance  of  finely  ground  glass  of  a  distinctly 
pale  green  tint. 

In  most  amoebae  a  nucleus  can  be  seen.  Its  detection 
is  not  always  possible  in  fresh  or  motile  amoebae,  but 
under  certain  conditions  in  the  motionless  or  dead 
amoebae  the  nucleus  becomes  evident,  and  it  may  be 
easily  shown  by  appropriate  staining  reagents.  It  is 
situated  eccentrically,  at  the  edge  of  the  entoplasm, 
and  appears  as  a  discoid  body,  about  6//  in  diameter, 
with  a  sharp  contour,  which,  though  occasionally 
broken  and  irregular,  is  generally  even;  it  may  be 
distinguished  from  vacuoles  of  the  same  size  by  its 
higher  refracting  power.  A  nucleolus  can  seldom  be 
observed,  and  in  stained  specimens  only. 

Foreign  bodies  are  frequently  seen  in  the  amoebae, 
especially  red  blood-cells.  These  are  sometimes  so 

41 


642  APPENDIX. 

numerous  that  the  whole  body  of  the  amoebae  is  filled 
with  them;  they  may  be  in  a  perfect  state  of  preserva- 
tion, or  quite  decolorized,  or  only  recognizable  by 
their  outline.  The  amoeba  rarely  contains  leucocytes 
or  fat-globules.  Various  forms  of  bacteria  are  more 
or  less  frequent  inclusions,  and  black  pigment  gran- 
ules and  irregular  brownish  masses  of  pigment  have 
been  noted  by  some  observers. 

Biological  Characters.  The  most  striking  and  char- 
acteristic feature  of  the  amoeba  is  its  motility.  This 
may  consist  either  in  an  alteration  of  its  shape  or  in 
an  actual  change  of  place.  Both  of  these  phenomena 
are  produced  through  the  mechanism  of  pseudopodia. 
These  latter  are  rounded,  blunt,  and  homogeneous 
processes  formed  by  the  more  or  less  gradual  protru- 
sion of  a  portion  of  the  ectoplasm  at  some  part  of  the 
periphery  of  the  amoeba.  The  motion  is  sometimes 
quite  gradual  and  continuous,  at  others  sudden  and 
jerky.  The  progressive  movement — that  is,  actual  loco- 
motion— is  brought  about  by  the  protrusion  of  pseudo- 
podia,  and  into  these,  when  they  have  reached  a  certain 
size,  the  granular  or  vacuolated  entoplasm,  with  its 
other  contents,  flows  with  a  more  rapid  movement 
than  that  by  which  the  pseudopodia  themselves  were 
formed.  Locomotion  is  generally  observed  to  take 
place  in  the  direction  of  least  resistance,  a  group  of 
cellular  elements  or  some  detritus  being  sufficient  to 
divert  the  course  of  the  amoeba.  The  amoeboid  move- 
ments are  also  influenced  by  various  factors,  particu- 
larly by  variations  of  temperature.  They  are  most 
active  at  the  mean  temperature  of  the  human  body, 
becoming  less  active  as  the  temperature  falls  or  rises 
above  this  mean.  They  become  motionless  in  a  tern- 


AMGEBA  COLL  643 

perature  lower  than  75°  F.  The  amoeba  does  not 
take  the  stain  of  various  coloring  solutions  until  the 
movements  cease,  presumably  on  the  death  of  the 
organism. 

Practically  nothing  is  known  of  the  conditions  of 
nutrition,  respiration,  and  reproduction  of  the  amoeba, 
as  no  observations  on  these  points  are  recorded. 

Occurrence  of  Amoebae  in  Man.  Amcebse  were  found 
in  the  stools  by  Kruse  and  Pasquale  in  forty  out  of 
fifty  cases  of  the  amoebic  type  of  dysentery;  by 
Kartulis  in  every  case  in  nearly  500  observations;  and 
by  Councilman  and  Lafleur  in  thirteen  out  of  fifteen 
cases;  while  in  their  remaining  two  cases  the  amoeba 
was  found  post-mortem,  either  in  the  material  scraped 
from  the  base  of  the  intestinal  ulcers  or  in  sections  of 
the  latter.  The  number  found  is  very  variable.  In 
some  cases  actively  moving  amoebae  are  found  in  great 
numbers  in  every  stool  examined  throughout  the  course 
of  the  illness,  while  in  other  cases  they  can  be  detected 
only  in  a  long  and  careful  search.  As  a  general  rule 
they  are  more  numerous  and  more  frequently  present 
in  the  acute  cases  or  in  the  earlier  stages  of  the  disease, 
or  in  the  periods  of  exacerbations  of  chronic  dysentery ; 
and  they  disappear  more  or  less  gradually  from  the 
stools  during  convalescence.  Occasionally  the  intestinal 
ulceration  is  latent,  the  motions  being  quite  formed, 
with  but  small  flakes  of  mucus  adherent  to  them,  in 
which  no  amoebae  may  be  found.  In  these  cases  the 
existence  of  dysentery  is  not  suspected  until  an  abscess 
of  the  liver  occurs  in  which  actively  motile  amoebae  are 
found,  either  by  exploratory  puncture  or  in  the  sputa 
if  the  abscess  evacuates  itself  spontaneously  through 
the  bronchi. 


644  APPENDIX. 

Numerous  investigations  have  demonstrated  conclu- 
sively that  amoebae  may  be  present  in  the  feces  of 
healthy  persons.  They  have  also  been  found  in  cases 
of  chronic  diarrhoea,  cholera,  intestinal  tuberculosis, 
typhoid  fever,  hemorrhoids,  and  other  diseases;  chiefly 
in  such  as  are  accompanied  by  looseness  of  the  bowels. 
Some  of  the  cases  cited  as  chronic  enteritis  or  chronic 
diarrhoea  were  in  all  probability  examples  of  the  more 
chronic  form  of  amoebic  dysentery,  but  not  all  of  them, 
of  course.  Temporary  looseness  of  the  bowels  in  other- 
wise healthy  persons,  either  as  the  result  of  slight  indis- 
position or  of  medication,  seems  to  be  a  condition  of  the 
presence  of  amoebae  in  the  stools.  Thus,  Schulberg 
found  these  organisms  in  ten  out  of  twenty  loose  stools 
produced  by  the  administration  of  Carlsbad  salts,  and 
concluded  that  the  amoeba  is  a  normal  and  harmless 
parasite  of  the  intestines,  the  reason  for  its  non-appear- 
ance in  ordinary  fecal  evacuations  being  the  solidity  and 
acid  reaction  of  the  contents  of  the  lower  bowel,  which 
soon  destroy  it.  The  question  naturally  arises  whether 
more  than  one  species  of  amoeba  is  found  in  the  human 
intestinal  tract.  So  far  no  definite  morphological  dif- 
ferences ha  ye  been  found  between  the  amoeba  occurring 
in  the  stools  of  healthy  persons  and  that  Jn  patients 
suffering  from  dysentery;  nor  can  any  deductions  be 
drawn  from  the  attempts  to  cultivate  the  amoeba,  for  no 
one  yet  has  succeeded  in  producing  pure  cultures  of  it. 

Pathogenesis.  It  is  evident  that,  in  the  absence  of 
artificially  produced  pure  cultures  of  amoebae,  inocula- 
tion experiments  must  be  made  wtih  material  such  as 
dysenteric  stools  or  the  contents  of  hepatic  abscesses. 
In  a  few  cases  such  material  from  hepatic  abscesses 
which  was  found  to  contain  no  organisms  other  than 


AMCEBA  COLL  645 

amoebae,  the  inoculations  have  been  made  in  three 
ways:  by  feeding  animals  with  material  containing  the 
amoeba,  by  inoculation  of  the  small  intestine  after  a 
preliminary  laparotomy,  and,  finally,  by  rectal  injec- 
tions with  or  without  suture  of  the  anal  orifice.  The 
first  method  has  always  proved  unsuccessful  except 
when  encysted  forms  were  present.  To  the  second 
method  the  objection  has  been  raised  that  the  manipu- 
lation of  the  intestines  and  the  use  of  antiseptic  solu- 
tions during  the  course  of  the  operation  are  in  them- 
selves a  source  of  irritation  to  the  bowel,  and  in  some 
cases  have  produced  an  enteritis.  The  third  method 
is  the  simplest,  and  has  given  positive  results  in  the 
hands  of  Losch,  Kruse,  Pasquale  and  others. 

The  results  of  the  last  two  observers  were  as  fol- 
lows :  Dysenteric  stools,  or  material  from  hepatic 
abscesses  containing  amoebae,  were  injected  into  the 
rectum  of  various  animals,  with  or  without  subsequent 
closure  of  the  anus,  for  twenty-four  or  forty-eight 
hours.  In  some  cases,  chiefly  those  in  which  motion- 
less amoebae  were  injected,  no  abnormal  result  followed; 
in  others,  blood-tinged  mucus,  containing  actively 
moving  amoebae,  appeared  in  the  evacuations  from  the 
second  day  or  thereabouts,  but  the  animals  did  not 
appear  to  be  ill;  in  a  third  series,  with  evacuations  of 
a  like  character,  the  animals  wasted  and  died  after 
a  variable  number  of  days.  In  both  the  second  and 
third  series  of  cases  post-mortem  examination  showed 
pathological  changes  in  the  large  intestine,  proportion- 
ate, as  a  rule,  to  the  severity  of  the  symptoms.  Of 
especial  interest  are  the  experiments  made  with  material 
from  liver  abscesses  which  were  proved  to  contain  no 
other  organism  than  the  amoeba.  Three  such  cases  are 


APPENDIX. 

recorded  in  cats,  in  all  of  which  an  experimental  dysen- 
tery was  produced.  The  lesions  found  are  reddening 
and  swelling  of  the  intestinal  mucosa,  chiefly  of  the 
lower  half  of  the  large  bowel,  with  here  and  there 
ecchymoses,  small,  superficial  areas  of  necrosis,  and 
shallow  ulcerations.  The  mesenteric  glands  and  the 
solitary  lymphoid  follicles  are  often  swollen.  In  the 
blood-tinged  mucus  covering  the  mucous  membranes 
amoebae  are  found  in  greater  or  less  numbers.  Micro- 
scopical examination  of  sections  of  the  intestine  shows 
that  the  necrosis  is  limited,  as  a  rule,  to  the  mucosa, 
and  that  beneath  it  the  submucosa  is  thickened  and 
oedematous  and  its  vessels  engorged;  there  is  also  small- 
celled  infiltration.  Amoebae  are  found  in  the  borders 
of  the  ulcers,  chiefly  in  the  follicles  of  Lieberkiihn; 
in  the  base  of  the  ulcers  they  rarely  penetrate  more 
deeply  than  the  upper  layers  of  the  submucosa.  With 
the  amoebae  are  found  many  bacteria,  chiefly  streptococci. 
From  a  comparison  with  the  lesions  of  amoebic  dysen- 
tery in  man  it  will  be  seen  that  while  the  processes  in  man 
and  in  the  cat  are  not  identical,  more  especially  as  re- 
gards the  depth  and  extent  of  the  ulceration,  yet  in  many 
points  the  resemblance  is  striking.  A  series  of  control 
experiments  was  undertaken,  by  the  authors  quoted, 
with  amoebae  from  the  stools  of  healthy  individuals  and 
the  straw-infusion  amoeba  of  Kartulis  obtained  in  his 
culture  experiments.  In  neither  of  these  cases  could 
an  experimental  dysentery  be  produced  in  any  of  the 
animals  inoculated.  They  conclude  that  it  is  proper 
to  designate  the  pathogenic  amoeba  as  the  amoeba  dys- 
enterice  (Councilman  and  Lafleur),  and  to  retain  the  name 
amoeba  coli  (Losch)  for  the  non-pathogenic  amoeba  of 
the  normal  healthy  intestine. 


AMCEBA  COLL  647 

Concerning  the  source  of  the  amoeba  and  the  mode  of 
infection  little  can  be'  positively  stated.  It  is  reason- 
able to  suppose,  however,  that  the  mouth  must  be  the 
usual  path  of  infection,  and  that  the  amoeba,  in  all 
probability,  is  taken  with  drinking  water. 

With  regard  to  other  organisms  found  in  amoebic 
dysentery,  a  great  number  of  bacteria  of  various  kinds 
and  some  flagellated  infusorial  organisms  are  associated 
with  the  amoeba  in  dysenteric  stools. 


CHAPTER  XXXIX. 

THE   MICRO-ORGANISM    OF    SMALLPOX    AND   COWPOX. 

No  bacteria  have  been  found  in  smallpox  which 
seem  to  have  any  relation  to  the  disease  except  as 
secondary  infections.  The  same  is  true  of  vaccinia. 
In  both  the  smallpox  and  vaccinia  papules,  vesicles, 
and  pustules,  L.  Pfeiffer  and  others  have  constantly 
found  small,  homogeneous  bodies  in  the  epithelial  cells 
surrounding  the  lesions.  These  little  bodies  are  in  the 
cell  substance,  not  in  the  nucleus,  and  usually  but  one 
or  two  exist  in  any  one  cell.  They  are  regularly 
missed  in  the  skin  when  vaccination  has  failed,  and 
also  in  similarly  appearing  papules  and  pustules  in 
pyaemia,  acne,  etc.  They  apparently  belong  to  the 
class  of  protozoa,  and  from  their  constant  presence  are 
believed  to  be  the  probable  specific  micro-organisms  of 
both  diseases.  They  are  at  first  about  double  the  size 
of  the  staphylococcus  and  increase  to  double  that  size 
(see  Fig.  87,  p.  651).  Similar  bodies  have  been  noted 
in  the  blood.  In  a  great  many  specimens  of  skin  from 
cases  of  variola  and  vaccinia  examined  by  Williams  in 
the  health  department  laboratories  these  bodies  have 
never  been  entirely  missed  in  the  epithelial  cells  sur- 
rounding the  lesions 

The  Connection  Between  Smallpox  and  Cowpox.  The 
inoculation  of  the  virus  of  smallpox  into  calves 
produces,  when  successful,  in  the  first  series  moderate 


SMALLPOX  AND  COWPOX.  649 

redness  and  swelling  at  the  point  of  inoculation,  with 
some  general  disturbance.  After  the  passage  through 
several  animals  an  affection  exactly  similar  to  cowpox 
occurs.  The  successful  inoculation  of  the  first  series 
of  cattle  from  smallpox  is  a  matter  of  great  difficulty, 
but  so  many  experimenters  have  asserted  that  they 
have  produced  lesions  similar  to  cowpox  from  small- 
pox that  there  seems  no  possibility  of  doubt  that  it 
has  been  done.  In  the  laboratory  we  have  failed  in 
several  attempts. 

Experiments  have  demonstrated  that  children  vacci- 
nated with  cowpox  vaccine  are  not  susceptible  to  inocu- 
lation with  smallpox  lymph,  and  also  that  those  who 
have  passed  through  smallpox  cannot  be  inoculated 
successfully  with  cowpox  vaccine.  The  mutual  immu- 
nity conferred  by  inoculation  with  either,  the  similar 
appearance  of  the  bodies  in  the  cells  about  the  vesicles 
of  both,  and  the  statements  from  reliable  sources  that 
smallpox  virus  has  produced  in  cattle  a  disease  indis- 
tinguishable from  cowpox,  leaves  hardly  any  doubt  that 
the  two  are  due  to  the  same  micro-organism,  which  has 
become  modified  by  transmission  through  cattle.  Why 
such  passage  should  produce  a  permanent  change  in  the 
virulence  of  the  organism  is  undoubtedly  a  difficult 
matter  to  explain,  but  we  must  remember  that  we 
know  practically  nothing  about  the  life-processes  of 
this  form  of  micro-organisms,  and  changes  once  pro- 
duced in  them  may  tend  to  become  fixed. 

The  Duration  of  the  Immunity  Conferred  by  Vaccina- 
tion. The  immunity  caused  by  successful  vaccination 
is  not  permanent,  and  varies  in  its  duration  in  different 
individuals.  Although  it  may  give  some  protection 
from  smallpox  for  ten  or  fifteen  years,  it  is  not  well 


650  APPENDIX. 

to  count  on  immunity  for  more  than  two  years,  and 
whenever  we  are  liable  to  exposure  it  is  well  to  be 
vaccinated.  If  it  was  unnecessary  it  will  not  be  suc- 
cessful, while  if  it  is  successful  we  have  reason  to 
believe  we  were  liable  to  at  least  a  mild  smallpox 
infection. 

Protective  Substances  Present  in  the  Serum  of  Animals 
After  Successful  Vaccination.  It  has  been  repeatedly 
shown  that  the  blood-serum  of  a  calf  several  weeks 
after  successful  vaccination  possesses  feeble  protective 
properties,  so  that  the  injection  of  one  to  two  litres  of 
it  into  a  susceptible  calf  would  prevent  a  successful 
vaccination.  A  further  and  more  convincing  fact  has 
been  demonstrated  in  the  laboratory  by  Huddleston — 
namely,  that  when  equal  parts  of  a  serum  taken  from 
a  calf,  two  weeks  after  successful  vaccination,  and  of 
an  active  vaccine  are  mixed  together  and  inoculated 
in  a  susceptible  calf  the  vaccine  fails  "to  take." 
The  serum  of  an  unvaccinated  calf  has  no  deleterious 
effect  whatever  when  mixed  with  the  vaccine.  Serum 
taken  even  several  years  after  vaccination,  if  the  case 
is  still  immune,  will  inhibit  very  distinctly  the  action 
of  fresh  vaccine  virus. 

The  Form  of  Vaccine  Virus  Used.  Vaccination  is 
now  usually  performed  with  calf  virus,  as  this  is  easier 
to  obtain,  is  just  as  reliable,  and  practically  eliminates 
the  slight  possibility  of  the  transferrence  of  syphilis 
which  existed  in  human  vaccine.  With  active  virus 
a  portion  of  skin  only  one- sixteenth  of  an  inch  in 
diameter  is  scratched  with  the  needle  and  the  virus 
rubbed  in.  If  preferred  it  may  be  inserted  by  a 
puncture.  The  vaccine  is  now  usually  mixed  with 
glycerin  and  water  and  placed  in  capillary  tubes.  So 


SMALLPOX  AND  GOWPOX. 


651 


prepared  it  is  much  more  durable  than  when  dried  on 
ivory  points  or  quills. 

The  Appearance  of  the  "  Vaccine  Organism."  Williams 
has  described  the  appearances  observed  as  follows  : 

"  The  epithelial  cells  of  the  skin  in  the  infected  areas 
of  vaccinated  calves  contain  characteristic  bodies.  These 
bodies  are  generally  spheroidal,  but  may  be  quite  irreg- 
ular in  outline.  They  vary  in  size  from  that  of  a 


FIG.  87. 


V    V 


Epithelial  cells  of  sebaceous  gland  of  hair  containing  vaccine  bodies. 
V.  Homogeneous  vaccine  bodies.    N.  Granular  nuclei.    X  about  600  diam. 


particle  of  nuclear  chromatin  to  that  of  the  nucleus 
iteelf,  and  are  sometimes  even  larger.  Usually  they 
are  about  one-fourth  the  size  of  the  nucleus.  They 
are  seen  only  in  the  body  of  the  cell,  never  in  the 
nucleus,  and  generally  only  one  is  seen  in  each  cell, 
though  there  may  be  three  or  four.  They  take  most 
nuclear  stains  rather  more  faintly  than  the  nucleus, 
and  are  distinguished,  moreover,  by  their  homogeneous 


652  APPENDIX. 

appearance.  With  hsematoxylin  (Dela field's)  and  eosin 
the  nucleus  of  the  epithelial  cells  take  an  irregularly 
granular,  dark  purple  stain,  while  the  peculiar  bodies 
are  a  fainter,  homogeneous  purple,  and  the  cell-bodies 
pink"  (Fig.  87). 

Horses,  rabbits,  and  sheep  were  successively  vaccin- 
ated with  calf  vaccine,  but  in  none  was  the  take  any- 
where as  good  as  in  calves,  nor  did  it  occur  in  every 
instance.  Guinea-pigs  and  dogs  failed  to  take  in  a 
few  trials.  The  pulp  and  serum  obtained  from  an 
epidemic  of  cowpox  took  feebly  in  calves  in  a  moder- 
ate percentage  of  those  inoculated.  The  characteristic 
vaccine  bodies  were  found  practically  identical  with 
those  in  vaccinia,  except  the  bodies  were  a  little  larger 
and  more  irregular  in  outline. 

The  Preparation  of  Vaccine.  For  the  following  sug- 
gestions I  am  indebted  to  Dr.  J.  H.  Huddleston,  who 
has  had  the  immediate  charge  of  the  production  of 
vaccine  for  the  New  York  Health  Department  for 
some  years : 

Seed  Virus.  A  sufficient  amount  of  vaccine  virus 
should  be  on  hand  to  vaccinate  forty  to  fifty  persons. 
Five  children  in  good  health,  and  not  previously  vac- 
cinated, should  then  be  vaccinated  each  in  a  spot  the 
size  of  a  ten-cent  piece.  On  the  fifth  day  after  vaccin- 
ation the  top  of  the  resulting  vesicle  should  be  removed 
and  sterilized  bone  slips  be  rubbed  on  the  base  exposed. 
It  should  be  possible  in  this  manner  to  charge  at  least 
from  one  to  two  hundred  slips  on  each  side  of  the  slip 
from  each  child.  The  slips  should  be  allowed  a  moment 
to  dry  and  then  placed  in  a  sterilized  box,  in  which,  if 
kept  in  cold  storage,  they  will  probably  remain  efficient 
at  least  two  or  three  weel^s. 


SMALLPOX  AND  COWPOX.  653 

Animals.  The  preferable  animals  are  female  calves, 
from  two  to  four  months  of  age,  in  good  condition  and 
free  from  any  skin  disease.  These  can  be  vaccinated 
on  the  posterior  abdomen  and  inside  of  the  thighs  easily 
by  placing  them  on  an  appropriate  table.  It  is  possible 
that  on  account  of  the  character  of  the  available  supply 
older  animals  may  be  desirable,  but  the  calves  take 
more  typically  and  are  more  easily  handled.  When 
an  animal  is  too  old  to  be  thrown  and  held  easily  it 
may  be  vaccinated  on  the  rump,  each  side  of  the  spine; 
but  the  skin  there  is  tougher,  and  the  resulting  virus, 
though  efficient,  is  not  so  easily  emulsified. 

Vaccination.  The  calf  should  be  cleaned  thoroughly, 
including  the  feet  and  the  tail,  and  the  hair  should  be 
clipped  from  the  end  of  the  tail.  The  posterior  abdo- 
men and  insides  of  the  thighs  are  then  shaved  and  the 
skin  beneath  washed  in  succession  with  soap  and  water, 
sterilized  water  and  alcohol,  and  then  dried  with  a 
sterile  towel.  On  this  area  there  are  then  made  about 
one  hundred  scarifications,  each  from  one-quarter  to 
one-half  of  an  inch  on  a  side.  The  scarification  is 
made  most  easily  by  cross-hatching  with  a  six-bladed 
instrument,  the  blades  being  about  one-thirtieth  of  an 
inch  apart.  The  scarification  is  superficial,  but  brings 
blood.  An  area  as  small  as  specified  is  less  likely  to 
become  infected  than  a  larger  one.  The  scarifications 
should  be  separated  from  each  other  by  an  interval  of 
at  least  one-half  to  three-quarters  of  an  inch.  After 
they  have  been  made  they  should  be  dried  with  a  sterile 
towel  or  cotton  and  rubbed  with  the  charged  slips.  One 
to  two  slips,  depending  on  the  amount  of  virus  each  slip 
contains,  should  be  sufficient  for  vaccinating  each  ves- 
icle. 


654  APPENDIX. 

Collection.  On  the  fifth  or  sixth  day,  depending  upon 
the  rate  of  development  of  the  vaccine  vesicles,  they 
should  be  ready  for  collection.  The  entire  shaved  area 
is  washed  with  sterile  water  and  sterile  cotton,  and  the 
crusts  are  picked  off.  The  soft,  pulpy,  remaining  mass 
is  then  curetted  off  with  an  ordinary  steel  curette  and 
the  pulp  placed  in  a  sterilized  vessel.  After  the  curet- 
tage  serum  exudes  from  the  torn  base  of  the  vesicle, 
and  ivory  slips  may  be  charged  in  this.  The  pulp 
should  be  mixed  with  from  two  to  three  times  its 
weight  of  glycerin  and  water,  equal  parts,  and  this  is 
done  most  effectively  by  passing  the  mixture  between 
the  rollers  of  a  Doring  mill.  A  watery  pulp,  especially 
if  it  is  not  to  be  used  immediately,  should  have  the 
smaller  proportion  of  glycerin.  The  emulsion  so  pro- 
duced can  then  be  put  up  for  issue  in  vials.  The  slips 
charged  with  the  serum  from  the  calf  may  also  be  used 
for  vaccinating.  Capillary  tubes  require  especial  means 
of  filling,  and  small  vials  filled  and  corked  answer  the 
purpose  admirably. 

Propagation.  Subsequent  animals  may  be  vaccinated 
in  any  one  of  the  three  ways:  (a)  Slips  may  be  charged 
from  typical  vesicles  on  primary  vaccinations,  just  as 
with  the  first  calf,  and  used  for  seed  virus;  (6)  slips 
charged  with  the  serum  from  the  calf  may  be  used  to 
vaccinate  a  second  calf;  (c)  the  glycerinated  emulsion 
may  be  used  to  vaccinate  succeeding  calves,  but  in  the 
last  case  it  is  necessary  to  keep  the  emulsion  a  varying 
length  of  time — often  two  or  three  months — before  it 
is  fit  for  use  to  vaccinate  the  calf,  because  the  use  of 
fresh  glycerinated  pulp  on  a  succession  of  calves  leads 
to  prompt  degeneration  of  the  vaccine  and  to  the  pro- 
duction of  infected  vesicles. 


SMALLPOX  AND  COWPOX.  655 

Laboratory.  The  laboratory  should  consist  of  at  least 
three  rooms:  (a)  Stable;  (6)  operating-room;  (c)  labor- 
atory-room. It  should  be  possible  to  make  and  keep 
all  the  rooms  clean.  The  stable  and  operating-room 
should  be  flushed  with  a  hose  and  hot  water  daily. 
Excreta  should  be  removed  immediately.  The  calves 
can  be  kept  cleaner  if  they  stand  on  a  raised  and  per- 
forated platform,  which  is  so  short  that  the  defecations 
cannot  fall  on  it,  and  if  they  have  no  bedding.  They 
must  be  fastened  to  keep  them  from  licking  the  scari- 
fications. If  they  are  fed  with  milk  the  dust  that 
would  be  imported  with  other  food  is  avoided.  In  the 
health  department,  when  a  calf  is  removed  its  stall  and 
platform  are  scoured  with  a  brush  and  sodium  carbonate 
solution.  The  stable  should  be  provided  with  a  shovel, 
broom,  hose,  currycomb,  mane  brush,  cord,  halters, 
and  with  buckets,  scrubbing  brushes,  and  sponges. 
The  operating-room  should  be  well  lighted  and  pro- 
vided with  a  table  and  stools. 

The  only  requisites  for  the  table  are  that  it  should 
be  heavy  and  firm;  that  it  should  have  holes  through 
the  top  so  arranged  that  straps  can  be  passed  through 
them  to  hold  the  calf  down,  and  a  vertical  strip  on  one 
side  of  the  table  to  which  the  upper  hind  leg  of  the  calf 
can  be  fastened.  The  calf  can  be  thrown  up  on  the 
table  easily  by  two  attendants. 

The  laboratory  should  also  be  well  lighted  and  fur- 
nished with  tables,  chairs,  desk,  case  for  instruments, 
and  refrigerator.  It  should  also  have  both  a  steam 
and  a  dry-air  sterilizer,  a  set  of  scales  weighing  to 
grammes  or  centigrammes,  and  a  blast  lamp  and  bel- 
lows. In  stock  there  should  be  one  to  two  thousand 
bone  slips  for  seed  virus,  and  ten  to  fifteen  thousand 


656  APPENDIX. 

smaller  slips  for  issue,  two  or  more  scarifiers,  a  curette, 
four  to  six  razors  for  shaving  the  animals,  a  razor  strop, 
a  pair  of  large  scissors,  curved  on  the  flat,  for  clipping 
the  animals,  a  burette  from  which  glycerin  flows  while 
the  vaccine  pulp  is  being  ground,  burette  holder,  a 
Dor  ing  vaccine  grinder,  clinical  thermometers,  to  take 
the  temperature  of  the  animals,  six  to  twelve  small 
glass  dishes  with  covers,  a  hard-rubber  syringe,  of  four 
ounce  capacity,  to  make  suction,  absorbent  cotton,  glass 
vials  and  corks,  and  several  pounds  of  soft  glass  tubing, 
three-eighths  of  an  inch  in  calibre,  to  store  virus  emul- 
sion. There  should  also  be  gowns  and  caps  for  the 
attendants.  Sodium  carbonate,  bichloride  of  mercury, 
bromine  (for  a  deodorizer),  alcohol,  and  glycerin  are 
the  chemicals  needed. 

For  issue  for  public  vaccinations  there  are  also  needed 
packing-boxes,  rubber  bands,  sheet  wadding,  needles, 
and  wooden  toothpicks  (for  removing  the  virus  from 
the  vials  and  rubbing  it  on  the  scarifications). 

Yield.  The  material  allowed  from  the  five  children 
should  vaccinate  at  least  five  calves;  it  may  easily 
vaccinate  fifteen  calves.  Ten  grammes  of  pulp  and  two 
hundred  charged  slips  would  be  an  average  yield  from 
a  calf,  and  that,  when  made  up,  should  suffice  to  vac- 
cinate at  least  fifteen  hundred  persons.  Calves  vary 
immensely  in  the  yield.  Of  two  calves  vaccinated  in 
precisely  the  same  way  one  may  furnish  material  for 
five  hundred  vaccinations  and  the  other  for  ten  thou- 
sand vaccinations. 

The  Durability  of  G-lycerinated  Virus  in  Sealed  Tubes. 
As  a  result  of  testing  from  time  to  time  an  immense 
number  of  specimens  of  vaccine,  the  conclusion  has 
been  reached  that  vaccine  properly  put  up  should 


SMALLPOX  AND  COWPOX.  657 

keep  at  least  three  months.  From  time  to  time  a 
single  lot  of  virus  will  fail  by  the  end  of  one  month. 
Sometimes  this  is  due  to  the  glycerin,  as  when  it  has 
some  chemical  impurity,  or  simply  that  the  glycerin  is 
not  diluted  sufficiently  with  water.  We  find  one  part 
of  water  to  two  of  glycerin  makes  a  good  dilution. 

Bacteria  in  Vaccine.  It  is  impossible  to  prepare  vac- 
cine so  that  it  is  at  the  time  of  its  removal  free  from 
bacteria.  In  fact,  there  are  usually  very  large  numbers 
of  one  or  more  varieties  of  bacteria  present.  When  the 
stable  and  animals  have  been  kept  clean  the  bacteria 
comprise  usually  very  few  varieties;  when  dirty  condi- 
tions prevail  the  bacterial  varieties  are  more  numerous. 
The  number  of  bacteria  found  varies  enormously.  The 
largest  number  found  by  us  was  126,360  in  one  loopful 
of  vaccine  virus,  and  the  smallest  number  523.  Discrete 
vesicles  at  the  borders  contain  many  less  bacteria  than 
the  confluent  ones  caused  by  the  inoculation  at  the 
scarification.  The  pulp  has  many  more  bacteria  than 
the  contents  of  the  vesicles.  The  period  which  elapses 
before  glycerinated  virus  becomes  sterile  is  also  quite 
variable,  but  does  not  depend  in  any  direct  way  upon 
the  number  of  bacteria  originally  present.  A  very 
large  number  may  disappear  rapidly,  and  a  few  persist 
for  a  long  time. 

After  two  or  three  weeks  the  number  of  living  bac- 
teria is  usually  greatly  diminished,  but  seldom  totally 
destroyed.  If  we  wait  until  the  vaccine  is  surely  sterile 
it  is  very  apt  to  be  also  useless — that  is,  the  vaccine 
bodies  are  dead  also. 

In  a  very  large  experience  we  have  learned  that  the 
number  of  bacteria  present  has  little  to  do  with  the 
resulting  vaccination.  The  character  of  the  vesicles  in 

42 


(558  APPENDIX. 

the  calves  and  the  trial  vaccinations  in  young  children 
give  a  much  more  reliable  basis  for  judging  of  the 
character  of  the  virus  than  any  bacteriological  counts 
of  colonies. 

Pathogenic  bacteria  other  than  the  practically  non- 
virulent  skin  staphylococci  are  not  found  when  animals 
are  properly  kept  and  vaccinated.  The  vaccine  pulp 
and  serum  mixture  is  added  to  two  and  one-half  to 
three  and  one-half  times  its  bulk  of  a  mixture  consisting 
of  two  parts  of  chemically  pure  glycerin  and  one  part 
of  water. 

Efficient  vaccine  should  be  inoculated  in  a  portion  of 
skin  no  more  than  one-eighth  inch  in  diameter. 

Care  of  the  Calves.  All  bedding  is  avoided  and  an 
exclusively  milk  diet  given;  thus  much  of  the  otherwise 
unavoidable  dust  is  done  away  with. 


CHAPTER   XL. 

RABIES  (HYDROPHOBIA). 

ALTHOUGH  neither  the  nature  of  the  micro-organism 
nor  the  nature  of  the  poison  of  rabies  has  as  yet  been 
determined,  it  is  here  considered  because  of  its  special 
interest,  and  from  the  fact  that  it  was  the  first  infec- 
tious disease  to  which  a  curative,  or,  rather,  preventive, 
method  of  inoculation  was  successfully  applied. 

Rabies  is  an  acute  disease  of  animals,  dependent  upon 
a  specific  virus,  and  communicated  by  inoculation  to 
man.  It  is  usually  associated  with  an  injury,  such  as 
the  bite  of  a  dog,  and  the  inoculation  of  the  broken 
surface  with  the  saliva  of  an  animal  affected  with  the 
disease.  This  is  the  so-called  rabies  of  the  streets. 
Wolves,  cats,  foxes,  and  dogs;  horses,  cows,  and  deer 
may  contract  the  disease;  monkeys,  rabbits,  and  guinea- 
pigs  are  all  inoculable  with  it,  as,  indeed,  are  all  warm- 
blooded animals.  Rabies  occurs  in  almost  all  parts  of 
the  world;  it  is  most  common  in  Russia,  France,  and 
Belgium;  it  is  not  infrequent  in  Austria  and  those  parts 
of  Germany  bordering  on  Russia,  and  in  England.  It 
is  comparatively  rare  in  this  country,  although  it  occurs 
occasionally  in  various  parts  of  the  United  States,  also 
in  Mexico  and  South  America.  Rabies  is  extremely 
rare  in  North  Germany,  Switzerland,  Holland,  and 
Denmark,  owing  to  the  wise  provision  that  all  dogs 
shall  be  muzzled;  and  in  Australia  it  is  unknown. 


660  APPENDIX. 

Etiology  and  Pathogenesis.  The  etiology  and  patho- 
genesis  of  rabies  are  still  but  imperfectly  understood. 
The  poison,  whatever  may  be  its  nature,  is  usually  con- 
tained in  the  saliva;  and  as  early  as  the  beginning  of 
this  century  experimental  rabies  was  produced  in  the 
dog  by  inoculation  with  the  saliva  of  a  hydrophobic 
patient.  The  bulk  of  the  toxic  material  appears  to  be 
excreted  in  the  saliva  of  the  parotid  gland,  though  a 
certain  small  quantity  may  be  excreted  by  the  other 
salivary  glands,  and  also  by  the  lachrymal  glands,  the 
pancreas,  and  the  mammse  of  rabid  animals.  The 
poison  may  also  be  found  in  the  suprarenal  bodies  and 
in  the  fluid  and  substance  of  the  cerebro-spinal  nervous 
system,  especially  the  medulla  oblongata;  it  is  found 
also  in  the  peripheral  nerves,  though  in  much  smaller 
quantity  than  in  the  central  nervous  system.  It  has  not 
been  found  in  the  blood,  the  urine  or  the  aqueous  humor 
of  the  eye;  it  has  been  reported  to  have  been  found  in 
the  foetus. 

That  the  disease  is  due  to  some  form  of  organism 
which  has  the  power  of  multiplying  in  the  tissues  and 
of  producing  a  toxic  substance,  which  appears  to  act 
specifically  upon  the  central  nervous  system,  cannot 
be  doubted.  As  in  other  specific  infectious  diseases, 
the  virus  is  transmitted  directly  from  animal  to  animal 
through  the  medium  of  some  fluid  or  secretion;  it  is 
now  very  generally  recognized  that  the  disease  cannot 
arise  anew,  as  was  at  one  time  assumed.  In  rabies, 
again,  as  in  other  infectious  diseases,  there  is  a  period  of 
incubation  during  which  the  poison  appears  to  increase 
in  quantity. 

The  certainty  with  which  the  disease  may  be  pro- 
duced and  its  severity  have  been  found  to  be  deter- 


RABIES.  661 

mined  by  three  factors  :  (1)  The  quantity  of  the  rabic 
virus  introduced;  (2)  the  point  of  inoculation;  (3)  the 
strength  of  the  virus  as  determined  by  the  kind  of  ani- 
mal which  affords  the  cultivation  ground  for  the  growth 
of  the  hypothetical  organism.  It  is  a  matter  of  com- 
mon observation  of  hydrophobia  in  man  that  slight 
wounds  of  the  skin,  of  the  limbs,  and  of  the  back  are 
often  followed  by  the  disease  after  an  extremely  long 
period  of  incubation;  while  in  lacerated  wounds  of  the 
tips  of  the  fingers,  where  small  nerves  are  numerous  or 
where  the  muscles  and  nerve-trunks  are  reached,  or  in 
lacerated  wounds  of  the  face,  where  there  is  a  similar 
abundance  of  nerves,  the  period  of  incubation  is  usually 
much  shorter  and  the  disease  generally  much  more 
rapid.  Experimental  infection  in  animals  is  produced 
with  the  greatest  certainty  when  the  material  from  the 
rabic  nerve-centre  (the  spinal  cord  or  bulb)  of  a  dog, 
or  of  a  human  being  who  dies  of  rabies,  is  injected  into 
the  dura  mater  of  the  brain.  It  may  be  produced  almost 
as  certainly  when  the  injection  is  made  into  the  anterior 
chamber  of  the  eye  or  into  the  greater  nerve-trunks. 
Intravenous  injection  is  usually  followed  by  positive 
results  in  small  animals,  but  the  larger  animals  do  not 
succumb  to  this  mode  of  inoculation.  Subcutaneous 
inoculation  in  animals  is  uncertain,  because  the  periph- 
eral nerves  are  not  always  injured;  but  injection  directly 
into  a  mass  of  muscle,  especially  into  parts  which  are 
rich  in  nerves,  almost  invariably  produces  the  disease. 
Absorption  of  the  rabic  poison,  even  from  a  healthy 
mucous  surface,  has  been  said  to  have  taken  place; 
and  the  conjunctiva,  the  nasal  and  genital  mucous 
membranes,  and  the  digestive  tract  have  been  noted  as 
unabraded  surfaces  from  which  this  has  occurred.  The 


662  APPENDIX. 

rapidity  with  which  the  virus  is  diffused  through  the 
body  from  the  point  of  inoculation  in  the  tissues  seems 
to  vary  according  to  the  location  of  the  wound,  but  it 
is  always  comparatively  slow.  It  has  been  found 
that  rabbits,  when  etherized  and  then  presented  to  a  mad 
dog  to  be  bitten  on  the  fur,  escape  the  disease  in  a  very 
large  proportion  of  cases,  although  the  teeth  may  have 
passed  well  through  the  skin;  if,  on  the  other  hand,  the 
part  presented  to  the  rabid  dog  be  shaved  before  it  is 
bitten,  the  bitten  animals  contract  rabies  in  a  much 
larger  proportion  of  cases.  So  in  man,  in  many  cases  the 
rabic  virus  may  be  cleaned  from  the  teeth  by  the  cloth- 
ing which  covers  the  bitten  part  before  they  come  in 
contact  with  the  skin.  From  what  has  been  said  it  is 
evident  also  that  when  the  skin  is  thick  and  the  nerves 
few  a  small  quantity  of  virus  may  find  its  way  into  a 
wound,  but  riot  penetrate  into  the  nerves,  and  thus  the 
person  bitten  by  a  rabid  animal  may  escape  without  any 
ill  effects  beyond  those  due  to  a  lacerated  wound.  This 
will  explain  the  fact  that  only  about  16  per  cent,  of 
the  cases  bitten  by  rabid  animals  appear  to  contract 
hydrophobia. 

Preventive  Inoculation  Against  Rabies.  The  old  treat- 
ment of  rabies  consisted  simply  in  encouraging  bleeding 
from  the  wound,  or  by  first  excising  the  wound  and 
then  encouraging  bleeding  by  means  of  ligatures,  warm 
bathing,  cupping-glasses,  etc. ;  the  raw  surface  was  then 
freely  cauterized  with  caustic  potash,  nitric  acid,  or  the 
actual  cautery.  It  is  doubtful  whether  the  disease  ever 
manifests  itself  after  such  heroic  treatment  if  the 
wound  be  small;  but  when  the  wounds  were  numerous 
or  extensive  the  mortality  from  it  was  still  high. 
As  it  was  often  impossible  to  apply  cauterization  to  the 


RABIES.  663 

wound  rapidly  or  deeply  enough  to  ensure  complete 
destruction  of  the  virus,  Pasteur  and  others  were  there- 
fore led  to  study  the  disease  experimentally  in  animals, 
with  the  hope  of  finding  some  method  of  immunization 
or  even  cure  through  bacteriological  methods;  these 
investigations  finally  resulted  in  the  discovery  of 
methods  of  preventive  inoculation  applicable  to  man. 

Immunization  against  rabies  may  be  effected  in  sev- 
eral different  ways.  Pasteur's  treatment  is  based  upon 
the  fact  that  rabic  virus  may  be  attenuated  or  intensified 
for  any  animal  at  will.  He  first  observed  that  the  tis- 
sues and  fluids  taken  from  rabid  animals  varied  con- 
siderably in  their  virulence.  Then  he  showed  that  the 
virus  taken  from  similar  positions — say  the  cerebro- 
spinal  fluid — had  always  the  same  action  in  the  same 
species;  but  that  fluid  taken  from  an  animal  of  different 
species  was  weaker  or  stronger  as  the  case  might  be. 
Thus  the  cerebro-spinal  fluid  of  a  series  of  dogs  is  of 
constant  strength,  and  inoculations  made  from  dog  to  dog 
regularly  produce  death  from  rabies,  the  animals  passing 
through  an  incubation  period  fairly  constant  in  length, 
and  through  a  series  of  similar  symptoms  up  to  death 
at  the  same  term.  If,  however,  a  series  of  monkeys 
be  inoculated  the  virus  gradually  becomes  attenuated, 
and  this  attenuation  becomes  more  and  more  marked  in 
successive  inoculations  until  eventually,  after  the  disease 
has  run  a  longer  and  longer  course  in  the  successive 
animals,  there  comes  a  time  at  which  the  virus  is  no 
longer  sufficiently  active  to  cause  death.  If  this  atten- 
uated fluid  be  now  passed  through  a  series  of  rabbits, 
dogs,  or  guinea-pigs  it  comes  back  to  such  a  strength 
that  it  will  kill,  though  slowly;  then,  however,  its  vir- 
ulence gradually  increases  until  the  original  intensity  is 


664  APPENDIX. 

reached.  If  successive  inoculations  be  made  into  rab- 
bits with  fluid,  either  from  the  dog  or  the  monkey,  the 
virulence  may  be  so  exalted  beyond  that  of  the  virus 
taken  from  a  street  dog,  in  which  the  incubation  period 
is  from  twelve  to  fourteen  days,  .that  at  the  end  of  the 
one  hundredth  passage  the  incubation  period  may  be 
reduced  to  about  six  or  seven  days.  This,  the  strong- 
est virus  obtainable,  was  called  by  Pasteur  the  fixed 
virus.  Rabic  virus  appears  also  to  become  attenuated 
under  certain  conditions  of  temperature,  and  if  it  be 
subjected  for  about  an  hour  to  a  temperature  of  50°  C. 
its  activity  is  completely  destroyed,  or  in  half  an  hour 
if  to  a  temperature  of  60°  C.  A  5  per  cent,  solution 
of  carbolic  acid,  acting  for  the  same  period,  exerts  a 
similar  effect,  as  do  likewise  1  : 1000  solutions  of 
bichloride  of  mercury,  acetic  acid,  or  potassium  per- 
manganate. The  virus  also  rapidly  loses  its  strength 
by  exposure  to  air,  especially  in  sunlight;  when  pro- 
tected from  heat,  light,  and  air  it  retains  its  virulence 
for  a  long  period.  In  his  earlier  experiments  Pasteur 
selected  a  series  of  rabic  poisons  of  different  strengths, 
beginning  with  that  obtained  from  the  spinal  cord  of 
the  monkey — from  the  very  weak  to  the  strongest  that 
he  could  obtain  in  this  animal — then  passing  through  a 
similar  series  obtained  during  the  process  of  exaltation 
of  the  virus  by  passage  through  the  rabbit.  By  inoc- 
ulating dogs  subcutaneously  with  virus  taken  from  a 
series  commencing  with  the  weakest  taken  from  a  mon- 
key, and  gradually  working  up  to  that  obtained  from 
the  rabbit — from  the  earliest  to  the  latest  in  the  series 
— the  animals  become  immune  not  only  against  subcu- 
taneous injection  but  against  subdural  injection  with 
fixed  virus,  and  also  against  the  bite  of  rabid  dogs. 


RABIES.  665 

Such  a  method  as  this,  however,  had  several  disadvan- 
tages, and  was  not  absolutely  certain  in  its  action,  as 
only  fifteen  out  of  twenty  dogs  were  completely  pro- 
tected. Pasteur,  therefore,  assisted  by  Chamberland 
and  Roux,  devised  a  more  trustworthy  and  accurate 
method,  in  which  he  utilized  the  fact  that  the  cord  of 
a  rabic  animal  when  kept  under  certain  conditions  loses 
its  virulence  in  fourteen  days.  A  series  of  cords  cut 
into  short  segments,  which  were  held  in  series  by  the 
dura  mater,  were  suspended  in  sterile  glass  flasks 
plugged  with  cotton  stoppers,  and  containing  a  quantity 
of  some  hygroscopic  material,  such  as  caustic  potash; 
and  the  whole  was  kept  at  a  temperature  of  about  22° 
C.  The  cord  when  taken  out  at  the  end  of  the  first 
twenty-four  hours  was  found  to  be  almost  as  active  as 
the  fresh  untreated  cord;  that  removed  at  the  end  of 
forty-eight  hours  was  slightly  less  active  than  that  re- 
moved twenty-four  hours  previously;  and  the  diminu- 
tion in  virulence,  though  gradual,  progressed  regularly 
and  surely  until,  as  already  noted,  at  the  end  of  the 
fourteenth  or  fifteenth  day  the  virus  was  inactive.  An 
emulsion  of  the  cord  of  the  last  day  was  made,  and  a 
certain  quantity  injected  into  a  dog  that  had  been  bit- 
ten; this  was  followed  by  an  injection  of  an  emulsion 
of  a  thirteenth-day  cord,  and  so  on  until  the  animal 
had  been  injected  with  a  perfectly  fresh  and,  therefore, 
extremely  active  cord,  corresponding  to  the  fixed  virus. 
Animals  treated  in  this  way  were  now  found  to  be  abso- 
lutely protected,  even  against  subdural  inoculation  with 
considerable  quantities  of  the  most  virulent  virus;  and 
thus  his  protective  inoculation  against  rabies  became  an 
accomplished  fact.  As  it  would  be  impossible,  how- 
ever, or  very  undesirable,  to  inject  any  but  persons  who 


666  APPENDIX. 

had  actually  been  bitten  by  a  rabid,  or  presumably 
rabid,  animal,  Pasteur  continued  his  experiments,  in 
order  to  see  whether  it  would  not  be  possible  to  cure  a 
patient  already  bitten.  He  carried  on,  therefore,  a 
series  of  experiments  which  led  to  the  discovery  that  if 
the  process  of  inoculation  be  begun  within  five  days  of 
the  bite  in  animals  in  which  the  incubation  period  was 
at  least  fourteen  days,  almost  every  animal  bitten  can 
be  saved;  and  that  even  if  the  treatment  be  com- 
menced at  a  longer  interval  after  the  bite  a  certain 
proportion  of  recoveries  can  be  obtained.  Thus  the 
application  of  this  method  of  treatment  to  the  human 
subject  was  not  tried  until  it  had  been  proved  in 
animals  that  such  protection  could  be  obtained,  and 
that  such  protection  would  last  for  at  least  two  years, 
and  probably  longer. 

The  chance  of  success  in  the  human  subject  appears 
to  be  even  greater  than  in  the  dog  or  rabbit,  seeing  that 
on  account  of  the  resistance  offered  by  the  human  tissues 
to  the  virus  the  period  of  incubation  is  comparatively 
prolonged;  very  rarely,  if  ever,  does  an  outbreak  of  the 
disease  in  man  occur  before  an  interval  of  at  least  fifteen 
days.  The  first  symptoms  usually  appear  in  the  fifth 
or  sixth  week,  sometimes  not  until  the  third  month  ; 
exceptionally  the  incubation  period  has  lasted  for  a 
year.  Thus  there  is  an  opportunity  of  obtaining 
immunity  by  beginning  the  process  of  vaccination 
soon  after  the  bite  has  been  inflicted,  the  protection 
being  complete  before  the  incubation  period  has  passed. 
In  his  earlier  experiments  Pasteur  injected  on  each 
succeeding  day  emulsions  from  a  cord  dried  for  one 
day  less  until  cords  dried  five  days  were  reached;  but 
later  he  used  those  dried  for  only  three  days.  This 


RABIES.  667 

was  the  u  simple"  ten-day  method.  It  was  soon  evident 
that  although  this  method  was  efficacious  where  the 
wounds  were  not  severe,  and  were  confined  to  parts  in 
which  the  nerve-supply  was  not  extensively  interfered 
with,  it  was  often  quite  inadequate  in  serious  cases, 
as  of  wounds  about  the  face,  or  of  wounds  inflicted 
by  a  mad  wolf,  the  virus  of  which  is  more  active  and 
the  lesions  made  more  severe  than  that  of  the  rabid 
dog  of  the  streets.  In  these  latter  cases  the  injec- 
tions which,  iu  the  simple  treatment,  are  spread  over 
five  days  are  made  in  three  days;  then,  on  the  four- 
teenth day,  a  fresh  series  of  injections,  or,  rather,  repe- 
titions, is  begun,  which  lasts  until  the  twenty-first  day. 
This  is  the  "  intensive  method."  In  the  technique  of 
the  treatment,  which  is  the  same  in  both  methods,  a 
small  portion  (about  1  cm.)  of  the  desiccated  cord  is 
rubbed  up  thoroughly  with  about  four  or  five  times  its 
bulk  of  bouillon  until  a  complete  emulsion  is  made; 
this,  then,  is  injected  by  means  of  a  syringe,  holding 
several  cubic  centimetres,  first  on  one  side  of  the  hypo- 
chondriac region  and  then  on  the  other,  the  following 
day,  and  so  on  alternately,  to  avoid  irritation.  With 
the  observance  of  thorough  asepsis  no  local  reaction  to 
speak  of  takes  place,  nor  are  abscesses  ever  formed. 
The  results  of  Pasteur's  method  of  protective  inocula- 
tion, as  recorded  in  the  reports  issued  in  the  Annales  de 
rinstitut  Pasteur  and  those  of  other  antirabic  institutes 
in  Italy,  Russia,  Roumania,  etc.,  are  very  favorable. 
Since  1886,  when  the  treatment  was  first  commenced  at 
the  Pasteur  Institute  in  Paris,  upward  of  20,000  per- 
sons bitten  by  rabid,  or  presumably  rabid,  animals  have 
received  preventive  inoculations,  with  a  mortality  of 
only  0.5  of  1  per  cent.  The  mortality  of  those  bitten 


668  APPENDIX. 

on  the  face  or  head  was  1.25  per  cent,  of  those  bitten  on 
the  hand;  it  was  0.75  of  1  per  cent,  of  those  bitten 
on  other  parts  of  the  body,  a  little  over  0.25  of  1  per 
cent.  As  a  rule,  only  those  persons  are  treated  who 
have  been  bitten  on  the  face  or  hands  or  whose  clothes 
have  been  lacerated,  so  that  the  virus  has  passed  into 
the  wounds.  Ordinarily,  a  certificate  from  a  physician 
or  a  veterinarian  that  the  animal  was  rabid  is  required 
before  vaccination;  but  if  the  animal  cannot  be  found 
or  the  wounds  are  severe  vaccination  is  performed  with- 
out it.  Taking  only  the  cases  in  which  rabies  has  been 
confirmed  in  the  animal  by  a  veterinary  surgeon,  the 
mortality  of  the  cases  treated  at  the  Pasteur  Institute 
in  Paris  is  only  0.6  per  cent. — a  proof,  it  would  seem, 
of  the  trustworthiness  of  the  statistics.  In  view  of  this 
fact  there  can  no  longer  be  any  doubt  of  the  value  of 
Pasteur's  antirabic  treatment.  It  has  been  stated  by 
some  that  the  percentage  of  persons  killed  by  the  bites 
of  rabid  animals  is  inconsiderable;  but  according  to  the 
reliable  statistics  of  Leblanc,  from  1878  to  1883,  out 
of  515  persons  bitten  in  Paris  83  died  of  hydrophobia, 
a  mortality  of  16  per  cent.;  most  authorities  place  the 
mortality  at  a  much  higher  figure.  Extensive  bites 
on  the  face  and  head  are  considered  to  be  particularly 
dangerous  ;  the  mortality  of  these  is  said  to  be  at  least 
80  per  cent.  The  bites  of  wolves  seem  to  be  more  fatal 
than  the  bites  of  dogs  or  other  animals;  the  mortality 
of  these,  in  spite  of  the  most  intensive  treatment,  is 
stated  to  be  still  10  per  cent.,  as  against  a  previous 
mortality,  without  specific  treatment,  of  40  to  60 
per  cent.  But  even  Pasteur's  antirabic  treatment 
appears  to  be  unavailable  when  symptoms  of  the  dis- 
ease have  manifested  themselves.  Our  results  in  the 


RABIES.  669 

New  York  Health  Department  have  been  very  en- 
couraging. 

Other  methods  of  immunization  against  rabies  have 
been  proposed  by  different  investigators.  But  all  of 
these  methods  have  proved  on  trial  to  be  unsatisfactory 
and  unreliable,  beside  being  not  devoid  of  danger.  As 
early  as  1889,  Babes  and  Lepp  conceived  the  idea  that 
it  might  be  possible  by  means  of  the  blood  to  transmit 
conferred  immunity  from  rabies  from  one  animal  to 
another;  but  although  the  success  of  these  investigators 
was  not  great,  Tizzoni  and  Schwartz,  and  later  Tizzoni 
and  Centanni,  worked  out  a  method  of  serum  inocula- 
tion and  protection  in  rabies  which  is  worthy  of  atten- 
tion. In  this  method  not  the  rabic  poison  itself  but 
the  protective  material  formed  is  injected  into  the 
tissues.  These  observers  showed  that  the  serum  of 
inoculated  animals  is  capable  of  destroying  the  patho- 
genic power  of  the  rabic  virus — not  only  when  mixed 
with  it  before  injection,  but  when  injected  simultane- 
ously or  within  twenty-four  hours  after  the  intro- 
duction of  the  virus  into  the  body.  This  serum 
treatment  of  rabies  is  still  in  the  experimental  stage. 
We  ourselves  have  had  no  experience  with  it,  nor  has 
it  been  adopted  in  Paris,  or,  so  far  as  we  know,  in  other 
places.  It  is  quite  possible  that  others  will  not  obtain 
such  good  results  as  the  authors  of  the  treatment,  or 
that  it  may  not  prove  so  efficacious  in  the  treatment  of 
man  as  it  has  been  found  to  be  in  experimental  work. 

The  Cauterization  of  Wounds  Infected  with  the  Virus 
of  Rabies  after  an  Interval  of  Twenty-four  Hours.  It 
is  commonly  believed  that  unless  a  cautery  is  used 
within  an  hour  after  infection  by  a  suspected  animal  it 
is  useless  to  apply  it.  This  belief  is  held  by  physi- 


670  APPENDIX. 

cians  in  general,  and  also,  apparently,  so  far  as  the 
literature  seen  by  me  indicates,  by  those  familiar  with 
rabies.  For  this  reason  physicians  when  applying  a 
cautery  later  than  an  hour  after  infection  do  so  largely 
as  a  matter  of  form,  for  its  moral  effect  on  the  patient, 
and  so  the  application  is  not  thorough,  and  in  conse- 
quence not  effectual.  There  is  no  evidence  to  show 
that  this  is  the  case  at  all;  no  systematic  investigations 
have  been  published,  so  far  as  we  know,  to  prove  the 
point  one  way  or  the  other. 

We  know  that  the  virus  of  rabies  is  not  carried  into  the 
system  by  the  blood,  but  through  the  nervous  system. 
Dr.  Follen  Cabot  carried  out  an  extensive  series  of 
experiments  in  the  laboratory  upon  guinea-pigs  which 
showed  :  1.  That  91  per  cent,  of  guinea-pigs  can  be 
prevented  from  developing  rabies  if  the  wounds  be  cau- 
terized with  chemically  pure  nitric  acid  at  the  end  of 
twenty-four  hours  from  the  time  of  infection,  probably 
a  larger  percentage  if  the  cautery  be  used  earlier.  2. 
That  fuming  nitric  acid  is  more  effectual  than  the  actual 
cautery  or  pure  nitrate  of  silver.  3.  That  some  degree 
of  benefit  is  derived  from  thoroughly  opening  and  swab- 
bing out  an  infected  wound  within  twenty  four  hours 
from  the  time  of  infection  when  no  cautery  is  used. 
I  believe  that  he  demonstrated  that  in  cases  in  which 
the  Pasteur  treatment  cannot  be  applied  great  benefit 
may  be  derived  from  the  correct  use  of  cauterization 
even  twenty-four  hours  after  infection,  and  that  even 
in  cases  in  which  the  Pasteur  treatment  can  be  given, 
an  early  cauterization  will  be  of  great  assistance  as  a 
routine  practice,  and  should  be  very  valuable,  as  the 
Pasteur  treatment  is  frequently  delayed  several  days, 
for  obvious  reasons,  and  does  not  always  protect.  In 


RABIES,  671 

the  case  of  small  wounds  all  the  treatment  probably 
indicated  will  be  thorough  cauterization  with  nitric 
acid  within  twelve  hours  from  the  time  of  infection. 
Our  experience  in  dealing  with  those  bitten  by  rabid 
animals  indicates  that  physicians  do  not  appreciate  the 
value  of  thorough  cauterization  of  the  infected  wounds. 
But  far  more  important  than  any  treatment,  curative 
or  preventive,  for  hydrophobia  in  man  is  the  prevention 
of  rabies  in  dogs,  through  which  this  disease  is  usually 
conveyed.  Were  all  dogs  under  legislative  control  and 
the  compulsory  wearing  of  muzzles  rigidly  enforced 
where  rabies  prevails,  hydrophobia  would  soon  become 
an  almost  unknown  disease.  This  fact  has  been  amply 
demonstrated  by  the  statistics  of  rabies  in  countries 
where  such  laws  are  now  in  force. 


INDEX  OF  INFECTIOUS  DISEASES  AND 
BACTERIA  FOUND  IN  THEM. 


Abscesses.  The  bacteria  most  commonly  found  in  acute  abscesses 
are :  Staphylococcus  pyogenes  aureus,  468,  and  albus,  470,  and  strep- 
tococcus pyogenes,  481-483.  The  following  species  are  also  occa- 
sionally met  with:  Pneumococcus,  509;  colon  bacillus,  449-453; 
typhoid  bacillus,  410;  micrococcus  tetragenus,  472,  and  influenza 
bacillus,  324.  In  "  cold  abscesses "  the  tubercle  bacillus  is  usually 
the  only  micro-organism  present.  Beside  these  bacteria  other  varie- 
ties may  sometimes  cause  circumscribed  suppurative  processes. 

Acne.  Unna  and  Hodara  (1894)  obtained  a  bacillus  from  the 
contents  of  acne  pustules  which  they  believe  to  be  the  cause  of  true 
acne  in  man.  Staphylococci  are  usually  present  in  the  pustules,  and 
undoubtedly  exert  some  influence  in  the  production  of  the  affection. 

Actinomycosis.  Due  to  the  presence  of  the  actinomyces  or  ray 
fungus,  618. 

Alopecia.  Although  many  dermatologists  consider  alopecia 
areata  to  be  of  neurotic  origin,  others  incline  to  the  belief  that 
this  affection  is  due  to  micro-organisms.  Definite  proof,  however, 
is  still  wanting  of  the  infectiousness  of  the  disease,  or  of  the  causal 
relation  of  any  specific  micro-organism  to  it.  Holborn  (1895)  de- 
scribed a  micra-ui'6..-  '  "  wlvrh  he  named  trichophyton  radeus, 
obtained  it  in  pufre  culture  ^  pi^aced  a  similar  affection  in  rab- 
bits by  inoculation,  claiming  that  it  was  the  cause  of  the  disease  in 
man. 

Angina.  When  not  diphtheritic  the  pyogenic  cocci,  483,  includ- 
ing the  pneumococcus,  are  most  frequently  found  in  angina,  also 
Vincent's  bacillus,  354. 

Anthrax.     Due  to  the  bacillus  anthracis,  554. 

Appendicitis.  In  thirty-two  put  of  thirty-five  cases  of  appen- 
dicitis bacteriologically  examined  by  Hodenpyl  (1893)  the  bacillus 

43 


674          INDEX  OF  INFECTIOUS  DISEASES. 

coli  communis,  451,  453,  was  the  only  micro-organism  present. 
Streptococci  and  other  varieties  of  bacteria  from  the  intestines  are 
also  occasionally  found. 

Arthritis.  The  pneumococcus  of  Fraenkel,  509,  has  been  fre- 
quently fuund  in  arthritis  following  pneumonia.  The  gonococcus 
of  Neisser,  528,  has  been  often  met  with  in  gonorrhceal  arthritis. 
Streptococci,  483,  and  staphylococci,  469,  have  been  obtained  from 
the  pus  of  the  affected  joints  in  suppurative  arthritis  following 
scarlet  fever. 

Beri-beri.  Various  species  of  bacteria  have  been  found  in  the 
blood  and  tissues  of  persons  affected  with  beri-beri,  but  none  of  these 
have  been  demonstrated  to  be  the  specific  cause  of  the  disease. 

Bronchitis.  From  the  sputa  of  patients  with  putrid  bronchitis 
a  spore-bearing  bacillus  has  been  obtained,  the  cultures  of  which 
gave  off  the  characteristic  odor  of  fetid  bronchitis.  Hitzig  (1895) 
obtained  two  bacilli  resembling  the  colon  bacillus  from  a  case  of 
putrid  bronchitis.  In  ordinary  acute  bronchitis  the  pneumococcus 
and  streptococcus  are  most  frequently  found,  but  also  small  cocci  like 
the  gonococci  in  shape,  and  occasionally  other  bacteria,  especially 
very  small  bacilli.  In  epidemics  of  influenza  the  influenza  bacillus 
is  frequently  found. 

Bronchopneumonia.  The  micro-organism  most  frequently  met 
with  in  bronchopneumonia  is  the  pneumococcus  of  Fraenkel,  507, 
508,  511 ;  next  to  this  the  streptococcus,  483  ;  then  FriedKinder's 
bacillus,  458,  and  the  staphylococcus — alone  or  in  combination. 
At  times  the  influenza  bacillus,  325,  is  often  found  also.  In  pneu- 
monia complicating  typhoid  fever  the  typhoid  bacillus  may  be 
present  in  almost  pure  culture. 

Bubo.  The  pus  from  an  unopened  inguinal  bubo  following  chan- 
croid is  usually  sterile,  though  it  may  sometimes  contain  the  ordinary 
pus  micrococci. 

Bubonic  Plague.  Due  to  the  presence  of  the  bacillus  pestis  of 
Kitasato  and  Yersin,  in  the  contents  of  the  buboes  and  in  the  blood 
of  infected  animals  and  man,  607. 

Carcinoma.  No  micro-organism  has  as  yet  been  demonstrated 
to  bear  any  causal  relation  to  cancer.  Some  attribute  the  disease  to 
protozoa. 

Cerebro -spinal  Meningitis.  The  micro-organism  most  fre- 
quently found  in  cerebro-spinal  meningitis  complicating  other  diseases 
is  the  pneumococcus,  510,  511,  of  Fraenkel  ;  while  in  uncomplicated 
epidemic  pases  the  diplococcus  intracellularis  raeningitidis,  516,  519, 


INDEX  OF  INFECTIOUS  DISEASES.  675 

of  Weichselbaum  is  usually  found.  Streptococcus,  483,  pyogenes  has 
also  been  met  with  in  a  certain  number  of  cases,  and  occasionally 
the  colon  and  typhoid  bacilli  and  other  species  of  bacteria. 

Chalazion.  Whether  stye  is  a  specific  affection  or  due  to  mixed 
infection  by  the  ordinary  pus  cocci  is  not  known. 

Chancroid.  Ducrey  (1890)  discovered  a  bacillus,  called  by  him 
bacillus  ulceris  cancrosi,  which  he  obtained  from  the  pus  of  soft 
chancres  and  buboes,  and  believed  to  be  the  cause  of  the  disease, 
but  he  and  others  who  have  found  it  failed  to  cultivate  it. 

Cholera  Asiatica.  Due  to  the  cholera  spirillum,  or  Koch's 
"comma  bacillus,"  579. 

Cholera  Infantum.  According  to  Booker  and  Jeffries,  Bagin- 
sky  and  others  cholera  infantum  is  not  due  to  a  specific  micro- 
organism, but  to  the  action  of  the  common  putrefactive  bacteria, 
such  as  the  colon  bacillus  and  the  proteus  vulgaris  and  other  allied 
species,  which,  decomposing  the  food  before  it  is  digested,  give  rise 
to  toxic  products,  which  are  then  absorbed  in  the  alimentary  canal. 

Cholera  Nostras.  Finkler  and  Prior  (1884)  obtained  from  the 
feces  of  patients  with  cholera  nostras  a  spirillum  which  they  be- 
lieved to  be  the  specific  cause  of  this  disease,  but  this  has  not  been 
corroborated  by  experiment  It  is  more  probable  that  cholera 
nostras,  summer  diarrhoea,  and  all  this  class  of  gastro-intestinal  dis- 
orders are  induced  by  the  development  of  toxic  products  as  the 
result  of  the  ferment  action  of  various  species  of  bacteria,  such  as  the 
colon,  452,  and  proteus  groups. 

Cholecystitis.  The  bacteria  most  commonly  found  are  the  colon 
bacillus,  453,  and  less  often  the  typhoid  bacillus  In  the  cases  where 
typhoid  infection  is  present  the  bacilli  may  remain  in  the  gall- 
bladder for  years. 

Cholelithiasis.  The  colon,  453,  and  less  often  typhoid  bacilli 
are  met  with.  Typhoid  bacilli  have  been  found  at  operations  for 
gallstones  ten  years  after  an  attack  of  typhoid  fever.  (See  Johns 
Hopkins  Hospital  Bulletin,  1899.) 

Conjunctivitis.  The  specific  infectious  forms  of  conjunctivitis 
are  undoubtedly  due  to  the  action  of  bacteria,  as  gonorrhreal,  525, 
ophthalmia,  and  perhaps  Egyptian  catarrhal  conjunctivitis  (to  the 
bacillus  discovered  by  Koch  and  studied  by  Kartulis,  Weeks  and 
others),  and  diphtheritic  conjunctivitis  (to  the  Klebs-LofHer  bacil- 
lus, 349,  when  associated  with  diphtheria,  or  perhaps  to  the  xerosis, 
348,  bacillus).  The  non-infectious  forms  of  conjunctivitis,  however, 
are  probably  due,  not  to  the  action  of  specific  micro-organisms,  but 


676  INDEX  OF  INFECTIOUS  DISEASES. 

rather  to  some  inflammation  resulting  from  any  cause  aggravated 
by  the  presence  of  pyogenic  micrococci. 

Coryza.  It  is  doubtful  whether  this  affection  is  due  to  the  action 
of  any  one  specific  micro-organism.  Bacteria,  however,  play  a  part 
in  keeping  up  the  inflammation  in  acute  and  chronic  nasal  catarrh  ; 
and  in  ozaena  the  offensive  odor  of  the  nasal  secretions  seems  to  be 
due  to  the  presence  of  certain  bacteria,  as  Hajek's  B.  foetidus  ozsense. 

Cystitis.  It  has  been  shown  by  recent  investigations  that  cyst- 
itis is  not  caused  by  the  mere  presence  of  most  varieties  of  bacteria 
in  the  bladder,  except,  perhaps,  by  a  few  varieties,  such  as  the  gono- 
coccus,  528,  provided  it  be  healthy ;  but  when  the  mucous  membrane 
is  injured  by  mechanical  violence,  or  by  the  presence  of  a  foreign 
body,  cystitis  is  likely  to  result  from  the  introduction  of  bacteria. 
The  micro-organisms  most  frequently  concerned  in  the  development 
of  chronic  cystitis  are  the  colon,  449,  bacillus,  the  typhoid  bacillus, 
the  bacillus  aerogenes,  and  varieties  of  the  proteus  bacillus.  Among 
other  bacteria  which  have  been  found  in  the  bladder,  and  which  may 
influence  the  production  of  chronic  inflammation,  are  the  tubercle 
bacillus,  staphylococcus  pyogenes  aureus  and  allied  species,  the  uro- 
bacillus,  and  the  urobacillus  liquefaciens  septicus  of  Krogius. 

Dengue.  No  specific  micro-organism  has  been  found  in  this  dis- 
ease which  would  seem  to  bear  a  causal  relation  to  it. 

Dental  Caries.  According  to  Miller  (1894),  who  has  made  an 
exhaustive  study  of  the  bacteriology  of  dental  caries,  it  is  a  mixed 
infection  due  to  the  presence  of  various  micro-organisms  in  the  pulp, 
cocci  and  bacilli  being  about  equally  frequent,  with  the  occasional 
appearance  of  spiral  forms.  The  typical  pyogenic  cocci  are  seldom 
present  in  the  pus  from  the  pulp,  but  various  allied  species  are 
found  which  cause  pus  formation  in  mice.  Putrefactive  processes, 
also  the  result  of  bacterial  action,  greatly  increase  the  action  of  the 
pulp  cocci. 

Diarrhoea.  The  action  of  bacteria  in  the  production  of  diarrhoea 
has  already  been  referred  to  under  cholera  infantum  and  cholera 
nostras  There  is  no  reason  to  suppose  that  any  particular  micro- 
organism is  the  specific  cause  of  this  class  of  diseases,  which  are  due 
probably  to  the  toxins  produced  by  various  bacteria.  Those  which 
would  seem  to  have  most  to  do  with  the  production  of  these  troubles 
are  bacilli  of  the  colon  and  proteus  groups. 

Diphtheria.  The  Klebs-Loffler  bacillus,  349,  is  now  recognized 
to  be  the  specific  cause  of  diphtheria.  Other  bacteria,  however,  are 
always  associated  with  this,  producing  more  or  less  of  mixed  infec- 


INDEX  OF  INFECTIOUS  DISEASES.  677 

tion — viz.,  the  streptococcus,  352,  staphylococcus,  353,  and  pneumo- 
coccus,  353,  mainly.  Rather  atypical  pseudomembranous  exudates 
are  also  produced  occasionally  by  the  pyogenic  cocci,  and  fairly 
characteristic  ones  by  the  fusiform  bacillus  of  Vincent,  354,  and 
probably  by  other  varieties. 

Distemper  in  Dogs.  According  to  Schantyr  (1893)  the  so- 
called  distemper  in  dogs  includes  three  different  infectious  diseases  : 

1,  abdominal  typhoid,  in  which  bacilli  closely  resembling  those  of 
typhoid  fever  in  man  are  found  in  the  blood  and  various  organs ; 

2,  dog-typhoid,  in  which  bacilli  are  present  which  are  readily  cul- 
tivated and  stain  by  Gram's  method  ;   and  3,  genuine  distemper, 
containing  bacilli  which  stain  by  Gram's  method,  but  which  do  not 
grow,  or  are  difficult  to  grow,  in  culture  media. 

Dysentery.  Trophic  or  amoebic  dysentery  is  probably  due,  in 
the  majority  of  cases,  to  the  presence  of  the  amreba  coli  found  in 
the  discharges.  But  this  parasite  has  not  been  found  in  all  forms 
of  dysentery  and  in  healthy  stools.  Among  other  bacteria  found 
in  the  alvine  discharges  which  may  be  concerned  in  the  etiology  of 
certain  cases  of  dysentery  are :  the  colon  bacillus,  the  proteus  bacil- 
lus, 542 ;  the  staphylococcus,  the  bacillus  pyocyaneus,  539  ;  the 
bacillus  dysenterica  liquefaciens,  etc. 

Eczema.  Various  species  of  bacteria,  micrococci  and  bacilli 
have  been  obtained  from  cases  of  eczema  seborrhoaicum  by  different 
investigators,  but  none  of  these  have  been  shown  to  be  specific  for 
the  affection. 

Empyenia.  The  streptococcus  pyogenes,  483,  is  the  usual  cause 
of  purulent  inflammation  of  the  pleura,  in  which  it  is  found  in  60 
per  cent,  of  cases.  Empyema  complicating  pneumonia  is  generally 
caused  by  the  pneumococcus  of  Fraenkel,  511  ;  and  tubercular  empy- 
ema  is  due,  of  course,  to  infection  by  the  tubercle  bacillus  The 
various  micro-organisms  are  often  found  together  in  the  same  case, 
with  one  or  the  other  predominating.  In  exceptional  cases  still 
other  varieties  of  bacteria,  as  the  typhoid  bacillus,  may  be  met  with. 

Endocarditis.  Numerous  varieties  of  bacteria  have  been  found 
in  pure  culture  or  mixed  in  cases  of  ulcerative  endocarditis,  the 
most  common  being  pneumococci,  509,  511,  streptococci,  483,  and 
staphylococci,  469  ;  more  rarely  gonococci,  529,  and  other  micro- 
cocci,  and  occasionally  bacilli  of  several  varieties,  are  found.  Most 
probably  the  action  of  bacteria  upon  the  endocardium  is  similar  to 
that  upon  the  bladder,  and  endocarditis,  like  cystitis,  is  not  usually 
produced  by  them,  unless  some  previous  injury  has  been  caused  to 


678  INDEX  OF  INFECTIOUS  DISEASES. 

the  valves,  when  their  introduction  then  increases  and  maintains  the 
inflammatory  process. 

Endometritis.  The  healthy  uterine  mucous  membrane  is  usually 
sterile,  but  various  species  of  bacteria  have  been  observed  in  the 
secretions  of  the  cervix  uteri.  In  inflammations  of  the  uterus  not 
following  abortion  or  child-birth  the  gonococcus  is  by  far  the  most 
frequent  micro-organism  found.  In  inflammation  following  child- 
birth and  operations  the  ordinary  pus  cocci  and  the  colon  bacillus 
are  also  frequently  met  with,  as  well  as  other  varieties  of  bacteria. 

Erysipelas.     Due  to  infection  by  streptococcus,  483. 

Fowl-cholera.  Due  to  infection  by  bacillus  cholerae  gallinarum 
(Fliigge),  probably  identical  with  the  bacillus  of  rabbit  septicaemia 
of  Koch. 

Furunculosis.  Due  to  infection  by  the  different  pus  cocci,  and 
more  especially  to  the  staphylococcus  pyogenes  aureus. 

Gangrene.  Etiology  not  positively  known,  but  probably  due  to 
the  invasion  of  various  parasitic  and  saprophytic  bacteria  into  the 
tissues  when  their  vital  resistance  has  become  lowered  by  malnu- 
trition and  pressure  or  by  a  poor  blood-supply. 

Gas-formation.  The  bacillus  aerogenes  capsulatus,  545,  has 
been  found  either  alone  or  along  with  pyogenic  bacteria. 

Glanders  or  Farcy.  Due  to  infection  by  bacillus  mallei,  600, 
602. 

Gonorrhoea.    Due  to  "gonococcus,"  528  (Neisser). 

Hog-cholera.  Due  to  infection  by  bacillus  of  hog-cholera  (Sal- 
mon and  Smith). 

Hog  erysipelas  or  Swine-plague.  Due  to  infection  by  bacillus 
of  swine-plague  (Salmon  and  Smith ) . 

Hydrophobia.  No  micro-organism  has  as  yet  been  discovered 
which  is  specific  for  this  disease,  660. 

Influenza.  La  Grippe.  Due  to  infection  by  the  bacillus  of 
influenza,  324.  Pneumococcus  inflammations  often  show  similar 
symptoms. 

Influenza  of  Horses.     Various  micro-organisms,  some  resem 
bling  the  pneumococcus  and  others  the  streptococcus  in  man,  have 
been  described  and  claimed  to  be  the  specific  cause  of  this  epidemic 
disease  in  horses. 

Keratitis.  According  to  Bach  (1895)  purulent  keratitis  is  due 
to  the  invasion  of  the  cornea  by  micro-organisms,  the  pyogenic  cocci, 
pneumococci,  etc.,  secondary  to  traumatism. 

Leprosy.     Due  to  the  bacillus  leprse,  316 


INDEX  OF  INFECTIOUS  DISEASES.  679 

Lupus.     Due  to  infection  by  the  tubercle  bacillus,  276. 

Lymphangitis.  Usually  due  to  streptococcus  pyogenes  ;  occa- 
sionally other  organisms — viz.,  staphylococcus  pyogenes  aureus  and 
albus  and  the  colon  bacillus,  either  alone  or  associated — take  part  in 
the  production  of  this  affection. 

Malaria.     Due  to  infection  by  the  plasmodium  malarise,  626. 

Malignant  (Edema.  Due  to  infection  by  bacillus  cedematis 
maligni. 

Malignant  Pustule.     Due  to  anthrax  bacillus. 

Mastitis.  The  micro-organisms  commonly  found  in  mastitis  are 
the  ordinary  pus  cocci — staphylococcus  and  streptococcus.  Diplo- 
cocci  corresponding  to  the  gonococcus  have  also  been  observed  in 
patients  suffering  at  the  same  time  from  gonorrhoea. 

Measles.  All  attempts  to  discover  the  etiology  of  measles,  as  of 
the  other  specific  eruptive  febrile  diseases  except,  perhaps,  smallpox, 
have  thus  far  been  futile. 

Meningitis.     (See  Cerebro-spinal  meningitis. ) 

Nephritis.  The  urine  in  acute  infectious  diseases,  and  also  in  cases 
of  chronic  nephritis,  not  infrequently  contains  various  micro-organ- 
isms, which  are  also  found  in  the  blood  or  some  other  of  the  organs. 
Among  the  micro-organisms  commonly  found  in  nephritis  second- 
ary to  general  infection  are :  Streptococcus,  staphylococcus,  pneu- 
mococcus,  bacillus  coli  communis,  bacillus  typhi  abdominalis,  etc. 

Ophthalmia.  There  can  be  little  doubt  that  most  acute  and  some 
chronic  inflammations  of  the  eye  are  due  to  the  presence  of  micro- 
organisms. As  is  well  known,  the  gonococcus  of  Neisser  is  the 
cause  of  gonorrhoeal  ophthalmia,  and,  according  to  Fuchs,  a  consid- 
erable number  of  cases  of  so-called  Egyptian  ophthalmia  are  probably 
due  to  the  same  infective  agent,  while  other  cases  are  perhaps  caused 
by  the  bacillus  of  Koch  and  Kartulis  or  by  a  combination  of  these 
two  micro-organisms.  Many  bacteria  when  introduced  into  the  eye 
give  rise  to  inflammatory  processes.  The  pneumococcus  has  been 
found  by  several  investigators  in  cases  of  panophthalmia  and  other 
metastatic  eye  affections,  sometimes  alone  or  associated  with  the 
streptococcus  and  staphylococcus  pyogenes  The  bacillus  pyocy- 
aneus  and  bacillus  coli  communis  have  also  been  met  with  in  these 
affections,  the  inflammation  being  undoubtedly  due  to  the  presence 
of  the  micro-organisms  in  the  eye,  which  has  been  previously  injured 
in  some  way. 

Osteomyelitis  and  Periostitis.  According  to  most  authors  the 
staphylococcus,  469,  pyogenes  aureus  is  considered  to  be  the  specific 


680          INDEX  OF  INFECTIOUS  DISEASES. 

cause  of  acute  osteomyelitis  ;  but  though  present  in  many  cases,  alone 
or  associated  with  other  bacteria,  this  is  not  the  only  organism  found 
in  the  affection.  Staphylococcus  pyogenes  albus,  streptococcus  pyo- 
genes,  pneumococcus,  and  bacillus  typhosus  have  also  been  found  in 
osteomyelitis  by  various  observers.  This  disease  cannot,  therefore, 
be  regarded  as  a  specific  infection,  but  is  rather  a  localized  infectious 
process  due  to  various  micro-organisms.  Chronic  osteomyelitis  and 
periostitis  may  also  be  considered  in  like  manner  as  localized  infec- 
tions due  to  the  tubercle  bacillus. 

Otitis  Media.  The  micro-organisms  most  frequently  found  in 
the  purulent  discharges  in  recent  cases  of  otitis  media  are :  Pneumo- 
coccus, streptococcus,  Staphylococcus  pyogenes  aureus  and  albus,  and 
Friedlander's  bacillus.  Occasionally  found  are  :  Bacillus  pyocyaneus, 
micrococcus  tetragenus,  bacillus  coli  com  munis,  and  diphtheria  ba- 
cillus, etc.  These  bacteria  are  undoubtedly  responsible,  directly  or 
indirectly,  for  the  inflammatory  process  and  pus  formation. 

Ozsena.  According  to  the  investigations  of  Babes,  Hajek  and 
others  the  micro-organisms  most  constantly  found  in  the  nasal 
secretions  of  this  affection  are  :  Friedlander's  bacillus,  or  a  capsule 
bacillus  closely  resembling  this,  and  the  bacillus  ozsense  of  Hajek, 
though  other  species  of  bacteria  are  also  often  present. 

Parotitis.  Simple  uncomplicated  mumps  is  probably  due  to 
some  specific  micro-organism  not  as  yet  discovered,  but  the  suppura- 
tive  inflammation  is  undoubtedly  caused  by  one  or  other  of  the 
ordinary  pyogenic  cocci.  In  parotitis  occurring  as  a  complication 
of  other  infectious  diseases,  as  pneumonia  and  typhoid  fever,  the 
specific  infective  agents  of  these  affections  have  been  obtained  in 
pure  culture  from  the  pus  of  the  parotid  abscess. 

Pericarditis.  Various  micro-organisms  have  been  found  in  the 
pericardial  sac  in  pericarditis— the  ordinary  pus  cocci,  pneumococci, 
bacillus  pyocyaneus,  tubercle  bacilli,  etc. 

Peritonitis.  Among  the  bacteria  found  commonly  in  peri- 
tonitis are :  The  ordinary  pyogenic  micrococci,  the  colon  bacillus, 
449,  453,  the  pneumococcus,  gonococcus,  typhoid  bacillus,  tubercle 
bacillus,  and  proteus  vulgaris.  The  pus  cocci,  especially  strepto- 
coccus and  the  colon  bacillus,  appear  to  be  the  usual  cause  of  the 
inflammatory  process  in  puerperal  peritonitis.  In  peritonitis  fol- 
lowing appendicitis  and  intestinal  injuries  the  colon  bacillus  is 
always  present  either  alone  or  associated  with  other  bacteria. 

Pleuritis.  Levy  (1895),  from  a  resume  of  the  literature  of  the 
subject,  arrives  at  the  conclusion  that  the  pneumococcus  is  the  usual 


INDEX  OF  INFECTIOUS  DISEASES.          681 

ause  of  pleurisy  in  children  and  of  metapneumonia  pleurisy,  but 
that  in  metastatic,  pyogenic,  pleuritic  inflammation  the  strepto- 
coccus or  staphylococcus  are  the  common  infective  agents.  Pleurisy 
due  to  streptococcus  or  staphylococcus  infection  is  not  in  all  cases 
attended  with  pus  formation  ;  the  exudate  in  a  certain  proportion  of 
cases  may  remain  serous. 

In  pleurisy  occurring  as  a  complication  of  typhoid  fever  the 
bacillus  typhosus  has  been  found  in  the  exudate.  Occasionally 
bacillus  coli  communis  has  been  found.  According  to  Flemming, 
about  41  per  cent,  of  the  fatal  cases  of  pleurisy  are  due  to  tubercular 
infection. 

Pleuropneumonia  of  Cattle.  Due  to  infection  by  the  pneumo- 
bacillus  liquefaciens  bovis  of  Arloing. 

Pneumonia.  Characteristic  lobar  pneumonia  is  due  to  infection 
by  the  pneumococcus,  507,  511  ;  irregular  cases  are  usually  due  to 
Friedliinder's  bacillus,  streptococcus,  staphylococcus,  typhoid  bacil- 
lus, and  influenza  bacillus,  325. 

Puerperal  Fever.  Due  usually  to  infection  by  streptococcus, 
482,  or  colon  bacillus,  453.  In  some  fatal  cases  staphylococcus 
pyogenes  aureus  has  also  been  found.  Among  other  micro-organisms 
sometimes  met  with,  and  which  in  these  cases  may  have  been  con- 
cerned in  the  production  of  the  inflammatory  process,  are  gonococcus 
and  proteus  vulgaris. 

Purpura  Hsemorrhagica.  No  micro-organism  has  been  shown 
to  be  specific  for  this  affection. 

Pyaemia.     (See  Septicaemia.) 

Pyelonephritis.  According  to  Schmidt  and  Aschoff  (1893), 
pyelonephritis  or  surgical  kidney  is  an  infectious  process  usually  due 
to  bacillus  coli  communis.  (See  also  Nephritis. ) 

Pyosalpinx  Zweifel  ( 1 892 )  has  shown  that  a  certain  proportion 
of  the  cases  of  pyosalpinx,  if  not  all  of  them,  are  due  to  the  pres- 
ence of  the  gonococcus,  528.  In  some  cases  the  infectious  agent  is 
apparently  streptococcus  pyogenes  or  pneumococcus ;  but  Zweifel 
believes  that  in  the  majority  of  cases  in  which  the  gonococcus  is  not 
found  it  is  the  infectious  agent,  its  absence  being  due  to  the  fact  that 
it  has  died  out  in  cases  examined  too  late  to  find  it. 

Relapsing  Fever.  Due  to  infection  by  spirillum  Obermeieri, 
596. 

Rheumatic  Fever.  The  close  analogy  existing  between  true 
rheumatism  and  certain  of  the  infectious  diseases,  such  as  gonorrhoaa, 
scarlet  fever,  and  septic  processes  in  general,  which  are  frequently 


682          INDEX  OF  INFECTIOUS  DISEASES. 

accompanied  by  arthritis  and  endocarditis,  has  led  to  the  belief  that 
acute  rheumatism  is  an  infectious  disease.  All  investigations  here- 
tofore made,  however,  have  failed  to  demonstrate  the  causal  relation 
of  the  different  bacilli  and  cocci  isolated  to  the  disease. 

Rhinitis  Fibrinosa.  Pseudomembranous  rhinitis  is  often  asso- 
ciated with  severe  faucial  diphtheria,  and  in  these  cases  virulent 
Klebs-Loffler  bacilli,  349,  are  present.  The  primary  form  of  the 
affection,  like  conjunctivitis,  usually  runs  a  favorable  course,  and  is 
due  usually  to  the  attenuated  diphtheria  bacillus  ;  but  here,  too, 
occasionally  virulent  diphtheria  bacilli  are  found  in  the  fibrinous 
exudate.  In  such  cases,  of  course,  the  nasal  infection,  however 
mild,  may  give  rise  to  severe  faucial  or  nasal  diphtheria  in  others. 
In  a  few  cases  only  pyogenic  cocci  have  been  found. 

Bihinosderoma.  A  localized  infectious  process  due,  apparently, 
to  the  presence  of  the  bacillus  of  rhinoscleroma. 

Rinderpest.  The  etiology  of  this  acute  exanthematous  disease 
in  cattle  is  still  obscure.  Recovery  from  an  attack,  however,  pro- 
duces marked  immunity,  and  Koch  has  achieved  considerable 
success  in  inoculating  cattle  against  rinderpest 

Scarlet  Fever.  Streptococci  are  constantly  present  in  large 
numbers  in  the  pseudomembranous  exudate  of  scarlatinal  angina, 
484,  and  not  infrequently  also  in  the  blood  and  organs  after  death 
from  scarlet  fever.  The  presence  of  these  streptococci  in  scarlet 
fever  is  probably  due  to  the  great  increase  in  the  streptococci  usually 
existing  in  the  throat  secretions,  and  does  not  indicate  any  specific 
causal  relation  to  the  disease. 

Septicaemia.  General  septicaemia  in  man  is  usually  due  to 
infection  by  one  or  other  of  the  common  pyogenic  cocci — strepto- 
coccus, 481,  483,  pyogenes  or  staphylococcus,  469,  aureus  and  albus. 
Other  micro-organisms  which  may  sometimes  be  concerned  in  the 
production  of  septicaemia  are  the  pneurnococcus,  509,  and  colon  ba- 
cillus. Septicaemia  in  cattle,  deer,  swine,  rabbits,  and  fowls  is  due 
to  infection  by  the  bacillus  of  fowl-cholera  or  rabbit  septicaemia 
specifically,  but  various  other  bacteria  produce  septicaemia  also  in 
rabbits,  mice,  swine,  and  fowls. 

Stomatitis.  Schimmelbusch,  Lingard,  Foote  and  others  have 
described  bacilli  obtained  by  them  from  the  necrotic  tissues  in  cases 
of  noma,  but  the  etiology  of  the  disease  is  by  no  means  established. 

Syphilis.  The  bacillus  of  Lustgarten,  309,  is  accepted  by  some 
to  be  probably  the  specific  cause  of  the  disease,  but  this  is  far  from 
proven. 


INDEX  OF  INFECTIOUS  DISEASES.  683 

Tetanus.     Due  to  infection  by  the  tetanus  bacillus,  388. 

Texas  Fever  in  Cattle.  Due  to  infection  by  a  blood  parasite 
belonging  to  the  protozoa,  described  by  Smith  under  the  name  of 
pyrosoma  bigeminum. 

Tonsillitis.     (See  Angina. ) 

Trachoma.  Various  micmcocci  have  been  found  in  trachoma 
by  different  investigators  and  claimed  by  them  to  cause  the  affection, 
According  to  Fuchs  and  Hoor,  trachoma  is  frequently,  if  not  always, 
due  to  infection  by  the  gonococcus 

Tuberculosis.  All  forms  of  tubercular  infection  in  man  and 
animals  are  due  to  the  bacillus  tuberculosis.  The  bacillus  which 
causes  tuberculosis  in  cattle,  299,  and  the  one  which  produces  it  in 
fowls,  300,  though  closely  resembling  the  tubercle  bacillus  in  man, 
possess  some  slight  differences. 

Typhoid  Fever.  Due  to  infection  by  bacillus  typhi  abdomi- 
nalis,  402. 

Typhus  Fever.  The  specific  causative  agent  of  this,  under 
certain  circumstances,  extremely  infectious  disease  has  not  yet  been 
determined. 

Varicella.  No  micro-organism  has  been  demonstrated  to  bear 
any  relation  to  the  etiology  of  this  affection. 

Variola  and  Vaccinia.  Probably  due  to  protozoa,  651.  The 
common  pus  cocci  and  various  other  micro-organisms  are  found  in 
the  characteristic  pustular  eruption  ;  their  presence  is  due  to  sec- 
ondary infection  of  the  pustules,  and  has  nothing  to  do  with  the 
cause  of  the  disease,  657 

Whooping-cough.  Considered  by  Koplik  and  others  to  be  due 
to  a  small  bacillus  found  in  the  nasal  and  bronchial  secretions  in 
cases  of  the  disease,  613. 

Wool-sorter's  Disease.     Due  to  anthrax  bacillus. 

Yellow  Fever.  Sanarelli  ( 1 897 )  discovered  a  small  bacillus,  609, 
in  the  blood  and  tissues  of  yellow  fever  cadavers,  which  he  named 
"bacillus  icteroides,"  and  claimed  to  be  the  specific  cause  of  yellow 
fever,  609. 


GENERAL  INDEX. 


CHORIONSchoenleinii  (favus  |  Antitoxins,   effect  of,    how   pro- 


fungus),  622 
Acids,  effect  of,  on  bacteria,  158 
formation    of,    from    alcohol, 

etc.,  83 

from  carbohydrates,  80 
Aerobic  bacteria,  141 
Agar,  nutrient,  215 

characteristic  of,  225 
Air,  bacteriological  examination 

of,  252 

Alexines,  120 
Amoeba  coli,  640 
dysenterise,  640 
Amoebae,  occurrence  of,  in  man, 

643 
Anaerobic  bacteria,  141 

culture  methods  for,  233 
Aniline  dyes,  166 

water  solution  of    fuchsin   or 

gentian  violet,  202 
Animals,  use   of,  for   diagnostic 

and  test  purposes,  236 
Anthrax  bacillus,  547 

biological  characters,  551 
growth  in  media,  552 
identification  of,  562 
infection,  how  caused,  559 
morphology  of,  548 
occurrence     in     cattle     and 

sheep,  556 
in  man,  557 
pathogenesis,  554 
spore  formation,  550 
infection,  prophylaxis  against, 

561 

Antiseptic  action,  151 
Antitoxins,  absorption  of,  taken 
by  the  mouth,  115 


duced,  363 
elimination  of,  by  the  body,  113 
nature  of,   362 

results  of,  in  treatment  of  diph- 
theria, 358 

stability  of,  in  the  serum,  113 
varieties  of  bacteria  producing, 

in  the  body,  113 
Apparatus,  sterilization  of,  222 
Arnold  steam  sterilizer,  213 
Arthrospores,  46 
Attenuation,  151 
Autoclave,  214 
Auto-infection.  131 


BACILLI,  general  characters  of, 
38 

Bacillus  aerogenes  capsulatus,  545 
anthracis,  547 

symptomatici,  563 
biology,  564 
morphology,  563 
pathogenesis,  564 
coli  communis,  444 
of  Friedlander,  458 
icteroides  (yellow  fever),  609 
of  malignant  oedema,  543 
mallei  (of  glanders),  598 
pestis  bubonicae  (plague),  606 
proteus  vulgaris,  539 

biological  characters,  540 
morphology,  539 
pathogenesis,  541 
pyocyaneus,  535 
biology,  535 
morphology,  535 
pathogenesis,  536 


686 


GENERAL  INDEX. 


Bacillus  of  tuberculosis,  263 
typhosus,  402 
of  whooping-cough,  613 
Bacteria,  adaptation  to  soil  of,  91 
attenuation  of,  262 
chemical  composition  of,  50 

effects  of,  60 
classification  of,  257 
definition  of,  34 
elimination  of,  from  the  body, 

115 

entrance  of,  into  the  blood,  116 
general  characteristics  of,  33 
influence  of,  one  species  on  an- 
other, 56,  137 
invasion    of,    in    tissues    after 

death,  198 

involution,  forms  of,  49 
manner  in  which  they  produce 

disease,  92 

morphological  forms  of,  35,  40 
motility  of,  58 
nutritive    substances    required 

for  growth  of,  52 
parasitic,  34,  89 
pathogenic,    presence    of,    on 

healthy  skin,  130 
of,  in  respiratory  tract,  130 
presence  of,  in  intestines,  131 
production  of  light  by,  59 
products  of,  general  symptoms 
caused     by     poisons     ab- 
sorbed, 93 
local  effects,  91 
relation  of,  to  disease,  85 
saprophytic,  34,  88 
size  and  nature  of,  33 
spore  formation  of,  46 
structure  of  cells  of,  42 
temperature  required  for  growth 

of,  56 

their  relation  to  oxygen,  54 
thermic  effects  of,  60 
vegetative  reproduction  of,  44 
Bacterial  species,  permanence  of, 

260  ^ 
Bactericidal  substances  in  blood  of 

immunized  animals,  108 
nature  of,  108 

Bedding,  carpets,  upholstery, etc., 
disinfection  of,  187 


Bichloride  of  mercury,  156 

solution  of,  170 

Blood,  bactericidal  effect  of,  119 
cultures,  how  obtained,  241 
-serum  coagulator,  220 
germicidal  effect  of,  119 
as  a  medium,  217,  268 
Books,  disinfection  of,  187 
Bouillon  nutrient,  212 
Bovine  tuberculosis,  299 

relation  of,  to  human  tuber- 
culosis, 299 
Bromine,  160 
Bubonic  plague  bacillus,  606 

immunizing     injections    in, 
608 


P<ALCIUM  compounds,  157 

\J     Camphor,  166 

Capaldi  plate  medium,  436 

Capsule  bacteria,  43 

Capsules  of  bacteria,  staining  of, 

203 

Carbolic  acid,  165 
solution,  170 
Carriages,    etc.,   disinfection   of, 

188 

Cerebro -spinal    meningitis,    bac- 
teriological diagnosis  of,  520 
Chemicals,  effects  of,  on  bacteria, 

151 
Chemotaxis,  negative,  121 

positive,  121 
Chlorine,  159 
Chloroform,  165 
Cholera  immunity,  581,  585 
infection,  how  caused,  582 
spirillum,  accidental  infection 

in  man,  579 
biological  characters  of,  570 

diagnosis  of,  587 
effect  of  chemical  disinfect- 
ants on,  576 

growth  in  gelatin  plate  cul- 
tures, 570 
stick  cultures,  572 
immunizing  injections  ( Haff- 

kine's),  585 
morphology,   569 
pathogenesis,  577 


GENERAL  INDEX. 


687 


Cholera  spirillum,  varieties  and 

variations,  585 
toxin,  580 
Cocci,  characters  of,  36 

staphylococcus     pyogenes    au- 

reus,  461 

streptococcus  pyogenes,  476 
Cold    predisposing   to   infection, 

130 

Colon  bacillus,  444 
biology  of,  445 
differential  diagnosis  from 

typhoid  bacillus,  454 
growth  on  common    media, 

446 

immunization,  451 
morphology  of,  444 
occurrence  in  man  and  ani- 
mals, 451 
pathogenesis,  448 
Copper  sulphate,  157 
Cover-glass     preparations,     how 

made,  198 
how  stained,  199 
-slips,  how  to  render  free  from 

grease,  198 
Cowpox,  micro-organisms  of,  648, 

651 

relation  to  smallpox,  648 
Creolin,  171 
Creosote,  166 
Cresol,  165 

Culture,  contamination  of,  231 
media,  reaction  of,  55 
method  of  making,  at  autopsy, 

241 
from  urine  and  feces,  243 


DENEKE'S  cheese   spirillum, 
592 

biology,  592 
morphology,  592 
Diphtheria      antitoxin,      actual 

steps  in  test  of,  373 
nature  of,  362 

of    its  action   on    toxin, 

363 

persistence  in  the  blood,  366 
production  of,  for  therapeutic 
purposes,  359 


Diphtheria  antitoxin,    technical 

points  in  testing,  366 
testing  of,  364 
unit,  definition  of,  366 
use  of,  in  treatment  and  im- 
munization, 365 
bacilli,    exudate  due   to,    con- 
trasted  with   that  due  to 
other  bacteria,  376 
persistence  of,  in  the  throat, 

350 
virulent,  in  healthy  throats, 

349 
bacillus,  329 

animal  inoculation  as  a  test 

of  virulence  of,  383 
biology  of,  337 
in   different  culture   media, 

336 

growth  on  agar,  338 
on  blood  serum,  338 
in  bouillon,  341 
history  of,  329 
isolation  of,  340 
morphology  of,  332 
non-virulent  forms,  347 
pathogenesis  of,  342 
staining  characteristics,  334 
toxin  of,  344 
bacteriological    diagnosis    of, 

377 
characteristic   appearances   of, 

375 

direct  microscopical   examina- 
tion of  exudate,  3  «2 
examination  of  cultures,  381 
mixed  infection  in,  352 
technique  of,  377 
toxin,  neutralizing  value  *of  a 

fatal  dose  'of,  369 
relation  between  the  toxicity 
and  neutralizing  value  of, 
366 

transmission  of,  355 
antitoxic  serum,  357 
susceptibility  and  immunity 

to,  356 
value  of  cultures  in  diagnosis 

of,  373 

Diphtheritic  inflammations,  loca- 
tion of,  347 


688 


GENERAL  INDEX. 


Diplococcus  intracellularis  men- 
ingitidis,  516  (See  Meningo- 
coccus^ 

pneumoniae,  498 
Disinfectants,  156-167 
testing  value  of,  152 
Disinfecting     chambers,     public 

steam,  190 
Disinfection,  152 
in   infectious    and    contagious 

diseases,  171 

practical   (house,    person,    in- 
struments, and  food),  168 
Dry-heat  sterilizer,  223 
Drying,  effect  of,  on  bacteria,  139 

FHRLICH'S  theory  of  the  pro- 

J-J  duction  of  antitoxin  in  blood, 
368 

Electricity,  influence  of,  on  bac- 
teria, 134 

Eisner's  method  for  cultivation 
of  typhoid  bacillus,  434 

Endospores,  46 

Epitoxoids,  Ehrlich's  theory  of, 
368 

Erlenmeyer  flask,  221 

Esmarch's  tubes,  228 

Essential  oils,  166 

l?ATS,  decomposition  of,  77 
T     Feces,  disinfection  of,  173 
Fermentation  tube,  82 
Ferments,  diastatic,  65 

enzymes,  63 

inverting,  65 

organized  and  unorganized,  62 

proteolytic  or  peptonizing,  64 

rennet,  65 
Flagella,  44 

staining  of,  205 
Formaldehyde,  160 

development  of,  from  trioxy- 
methylene  pastilles,  184 

forms  of  apparatus  for  develop- 
ing, 181 

gas,   advantages  of,  over  sul- 
phur dioxide,  188 

and  sulphur  dioxide  gases, prac 
tical  employment  of,  178 


Formalin,  170 

Friedlander's  bacillus,  458 

biological  characters  of,  458 
morphology  of,  458 
pathogenesis  of,  459 

Fungi,  pathogenic  varieties,  619 


riABBETT'S  solution,  304 
iJ     Gas,  formation  of,  from  car- 
bohydrates, 81 
Gelatin,  nutrient,  215 

characteristics  of,  225 
Germination  of  spores,  49 
Glanders  bacillus,  598 
isolation  of,  603 
morphology,  598 
pathogenesis,  600 
test  for  (mallein),  604 
Gonococcus   in  cases  of   chronic 

urethritis,  533 

Gram's  method  of  staining,  532 
Neisser,  522 

biological  characters  of,  525 
morphological  characters  of, 

522 
in  pus-cells,  524 

affections  due  to,  528 
immunizing  serum  for,  529 
pathogenesis,  529 
Gonorrhoea,  bacteriological  diag- 
nosis of,  530 
Gram's  method  of  staining,  203 


TJAFFKINE'S    preventive   in- 

11     oculation  for  cholera,  585 
for  plague,  609 

Hands,  disinfection  of,  192 

Hanging  drop  for  study  of  bac- 
teria, 209 

Heat,  dry,  effect  of,  on  bacteria, 

147 
moist,  effect  of,  on  bacteria,  147 

Hiss'  media  for  typhoid  bacillus, 
430 

Hydrogen  peroxide,  159 

Hydrophobia,  659 
etiology  of,  660 

preventive    inoculation    (Pas- 
teur), 662 


GENERAL  INDEX. 


689 


Hydrophobia,    preventive,    cau-    Koch's  tuberculin,  288 


terization  of  wounds,  669 

IMMUNITY,  acquired,  to  poi- 
1       son,  112 
to  bacterial  poisons,  123 
decrease  of,  103 
natural,  102 
produced  by  injection  of  serum 

of  immunine  animals,  109 
specific,  106 

how  acquired,  107 
theories  of,  119 
Immunization     by     non-specific 

means,  104 
Incubators,  231 
Infected    dwellings,    disinfection 

of,  186 
Infection,  influence   of  quantity 

in,  95 
inherited  and  susceptibility  to 

or  immunity  from,  132 
mixed,  98 

modes  of  entrance  of,  100 
removal  by  local  means,  105 
spread  of,  128 
theories  of,  123 
Influenza  bacillus,  320 

biological  characters,  321 
detection  of,  in  sputum,  322 
immunity  to,  325 
morphology  of,  321 
pathogenesis  of,  324 
pneumonia  from,  325 
resistance  of,  to  drying,  323 
staining    characteristics    of, 

321 

in  tuberculosis,  326 
diagnosis  of,  327 
spread  of,  327 

Instruments,   dressings,  etc.,  for 
surgical  operation,  disinfection 
of,  191 
Iodine,  160 

trichloride,  160 
lodoform,  165 
Iron  sulphate,  157 


KLEBS-LOFFLER    bacillus, 
329 


old  tuberculin,  289 

1  EPROSY  bacillus,  315 
Lj     biological  characters  of,  316 
differential  diagnosis  of,  318 
location  of,  in  tissues,  316 
morphology  of,  315 
pathogenesis  of,  316 
direct  inheritance  of,  317 
Ligatures,  disinfection  of,  192' 
Light,  influence  of,  on  bacteria, 

135 
Lime,  chloride  of,  160,  170 

milk  of,  170 

Loffler's  alkaline  solution  of  me- 
thylene-blue  for  staining  diph- 
theria bacillus,  334 
Lupus,  278 

Lustgarten's  bacillus,  311 
Lysol,  171 

MALARIA,  diagnosis  of,  635 
infection,  how  acquired,  638 
mixed  infection  in,  635 
technique  of  blood  examina- 
tion in,  636 
Malarial  parasites.    See  Plasmo- 

dium  malarise,  626 
Material  for  bacteriological  ex- 
amination, procuring  of,  from 

those    suffering   from    disease, 

240 

Mallein  test  for  glanders,  604 
Media  to  be  used  at  autopsy,  241 

sterilization  of,  218 
Meningococcus,  516 

biological  characters  of,  517 

morphology  of,  516 

pathogenesis,  518 
Mercury,  bichloride,  156 

biniodide,  157 
Mesophilic  bacteria,  145 
Metachromatic  bodies,  42 
Metbylene-blue  solution,  202 
Metschnikoflf's  theory,  122 
Micrococcus  gonorrhoeae,  522 

lanceolatus,  498 

tetragenus,  472 
Microscope,  different  parts  of,  206 


44 


690 


GENERAL  INDEX. 


Microscopical  examination  of  un- 
stained bacteria,  198 
Migula,  classification  of  bacteria 
/^  by,  258 

/  Milk  as  a  culture  medium,  216 
(          sterilization  of,  193 
\.       streptococci  and  staphylococci 
^    in,  117 

tubercle  bacilli  in,  117,  283 
Mixed  infection,  137 

examination    for  other  bac- 
teria in  tuberculosis,  305 
practical  notes  in  the  exami- 
nation for,  307 

Mosquitoes  as  agents  of  infection 
in  malaria,  638 


NEISSER'S  stain  for  diphtheria 
bacillus,  335 
Nitrogen   combination   produced 

by  bacteria,  79 
Nuclein,  121 


PARASITES,  strict,  91 

I      Pasteurization,  149 

Pasteur's  flask,  221 

Petri  dish,  225 

Pfeiffer's  cholera  reaction,  582 

Phagocytosis,  122,  125 

Phenolphthalein  as  an  indicator, 

218 
Pigmented  leucocytes  in  malaria, 

632 

Pigment  production,  66 
Plasmodium  malaria?,  626 

sestivo    autumnal      parasite, 
630 

effect  of  quinine  upon,  634 

inoculation      experiments 
with,  633 

quartan  parasite,  629 

tertian  parasite,  627 
Plasmolysis,  42 
Plate  cultures,  225 

study  of  colonies  in,  228 
of,  in  gelatin,  230 

surface  inoculation  of,  228 

technique  of  making   224 
Pneumococcus,  498 


Pneumococcus,    attenuation     of 

virulence  of,  506 
biological  characters  of,  500 
in  blood,  510 
diseases  caused  by,  511 
effects    of    germicidal    agents, 

light,  and  drying  on,  503 
growth  on  ordinary  media,  500 

on  special  media,  502 
immunity  to  infection  by,  513 
morphology  of,  498 
occurrence  in  man,  507 
pathogenesis  of,  504 
producing  mixed  infection,  508 
source  of  infection  by,  504 
varieties  of,  511 

Potatoes  as  medium,  216 

Proteus,  71 

Pseudodiphtheria  bacilli,  351 

Pseudomembranous  inflamma- 
tions due  to  bacteria  other  than 
the  diphtheria  bacilli,  353 

Pseudotuberculosis,  streptothrix 
of,  619 

Psychrophilic  bacteria,  145 

Ptomai'ns  and  toxins,  68 

Pure  cultures,  230 

Putrefaction,  78 

Pyogenic  cocci,  461,  476 


RABIES,  659 
cauterization     of      infected 

wounds,  669 
Reaction  in  media,  correction  of, 

217 

Reduction  processes,  75 
Rooms,  disinfection  of,  175 

OAPROPHYTES,    facultative, 
O     90 

strict,  90 

Scarlet  fever,  streptococci  in,  354 
Sections,  preparation  of,  200 
Serum,  anti-pneumococcic,  514 
treatment  of  pneumonia  by, 

514 

antistreptococcic,  489 
preparation  of,  492 
protection  afforded  by,  491 


GENERAL  INDEX. 


691 


Serum  antistreptococcic,  stability 

of,  492 
standardization  of.  value  of, 

492 

therapeutic  results  of,  493 
protective,  test  of,  110 
test,  417 

Silver,  nitrate,  157 
Skin,  disinfection  of,  192 
Smallpox,    micro-organisms    of, 

648 

Smegma  bacillus,  313 
Sodium  compounds,  157 
Specific  immunity,  nature  of,  124 
Spirilla,  characters  of,  39 
Spirillum  of  Asiatic  cholera.   See 

Cholera,  568 
Finkler  and  Prior's,  589 
biology,  589 
morphology,  589 
pathogenesis,  591 
Metschnikovi,  593 

biological  characters  of,  594 
Obermeieri,  596 

in  relapsing  fever,  596 
Sporagenous  granules,  42 
Spores,  staining  of,  203 
Spore  types,  48 
Sputum,  collection  of   material, 

301 

from  consumptive  patients,  dis- 
infection of,  174 
general  rules  in  microscopical 

examination  of,  308 
methods    of    examination    for 

tubercle  bacilli  in,  302 
washing  of,  306 

Staining  solutions,  ordinary,  200 
Staphylococcus  epidermis  albus, 

471 

pyogenes  albus,  471 
aureus,  461 
biology,  461 
growth  in  common  media, 

462 

immunization,  467 
morphology,  461 
occurrence  in  man,  468 
pathogenesis  of,  464 
production  of  toxic  sub- 
stances by,  466 


Staphylococcus  pyogenes  citreus, 

472 
Sterilization,  fractional,  149 

incomplete,  152 
Sternberg  bulb,  251 
Streptococci,  dettction  of,  in  pus, 

etc.,  496 
of,  in  blood,  496 
Streptococcus  erysipelatis,  476 
pyogenes,  476 

biological  characters  of,  478 
growth  on  media,  478 
immunity,  487 
morphology  of,  477 
occurrence  of,  in  man,  483 
pathogenesis,  481 
toxins  of,  in  sarcoma,  485 
production     of     toxic     sub- 
stances by,  487 
susceptibility  to  infection  by, 

487 
use  of  anti-streptococcic  serum, 

489 

Strep  tothrix,  615,  619 
actinomyces,  61 K 
in  pseudotuberculosis,  619 
Sulphur  dioxide  gas,  158 

in  house  disinfection,  189 
Sulphuretted  hydrogen,   produc- 
tion of,  74 

Sunlight,  effect  of,  168 
Sweat,    bacteria    eliminated   by, 

117 
Syphilis  bacillus,  311 

biological     and    pathogenic 

properties  of,  312 
morphology  of,  311 


TEMPERATURE,  effect  of,  on 
bacteria.  146 
Tetanus  antitoxin,  395 
dosage,  400 

persistence  in  the  blood,  398 
production  of,  for  therapeu- 
tic purposes,  397 
results  in  treatment  in  teta- 
nus, 399 

technique    of    testing    anti- 
toxin serum,  307 
bacillus,  385 


692 


GENERAL  INDEX. 


Tetanus  bacillus,  biology  of,  386 
differential  diagnosis  of,  400 
morphology  of,  385 
non-virulent  type,  401 
pathogenesis,  388 
resistance  of  spores,  388 
toxin,  392 

action  of,  in  the  body,  393 
varieties  of,  due  to  bacillus,  391 
Tetragenus,  micrococcus,  472 
biology  of,  474 
growth  of,  on  media,  474 
morphology  of,  473 
pathogenesis  of,  474 
Thermo-regulator,  232 
Thermophilic  bacteria,  145 
Timothy  grass  bacilli  and  differ- 
ential diagnosis  from  tubercle 
bacilli,  319 
Toxalbumins,  71,  72 
Toxins,  73 

Trichophyton     (ringworm     fun- 
gus), 620 

Tricresol,  166,  171 
Tube    cultures,    inoculation    of, 

from  plate  colonies,  229 
Tubercle  bacillus,  263 

action    of,    upon    tissues  of 

poisons,  277 
attenuation  of,  287 
biological  characters  of,  265 
distribution  of,  in  tissues,  278 
duration  of  virulence  of,  in 

sputum,  286 

effect  of  sunlight  on,  267 
growth  on  coagulated  blood- 
serum,  268 
on  peptonized  veal  or  in 

beef  broth,  269 
mode  of  infection,  278 
morphological  characters  in, 

264 

pathogenesis  of,  273 
paths  of  infection,  278 
practical  examination  for,  in 

sputa,  301 
resisting  power  of,    against 

disinfectants,  266 
smegma  bacillus,  and  leprosy 
bacillus,  differential  diag- 
nosis of,  314 


Tubercle  bacillus  from  sputa  and 

infected  materials,  269 
staining  of,  in  tissues,  310 
a  strict  parasite,  267 
transmissibilily  of,  to  foetus, 

285 

Tuberculin  for  diagnosis  of  tuber- 
culosis in  cattle,  291 
in  man,  294 
Tuberculosis,    animals    attacked 

by,  276 

antituberculous  serum  in,  297 
auto-infection    by    swallowing 

sputum,  284 
communicability  of,  263 
different  forms  of,  in  man,  276 
immunization  in,  288 
individual    susceptibility    to, 

281 

infection  from  coughing,  sneez- 
ing, etc.,  283 
by    ingestion    of    milk    and 

meat,  283 
inhalation  of  tuberculous  dust, 

279 

mixed  infection  in,  287 
prophylaxis  of,  298 
Turpentine,  oil  of,  166 
Typhoid  bacillus,  402 

biological  characters,  405   v' 
comparative  value  of  methods 

for  isolation  of,  439 
detection  of,  in  water,  443 
differential    diagnosis    from 

colon  bacillus,  454 
fermentation,  407 
flagella,  404 
growth  in  bouillon,  406 
on  gelatin  plates,  405 
in  milk,  407 
on  potato,  406 
indol  reaction,  407 
infection,    diagnosis    of,    by 

Widal  test,  416 
isolation  of,  from  feces,  urine, 

blood,  water,  etc..  429 
length  of  time  capable  of  liv- 
ing outside  the  body,  413 
location  of,  in   body  in  ty- 
phoid fever,  40'J 
methods  of  infection,  412 


GENERAL  INDEX. 


693 


Typhoid  bacillus,  morphological 

characters  of,  403 
in  oysters,  413 
pathogenic  properties  of,  407 
in  pneumonia,  410 
presence  of,  in  blood,  411 

in  feces  and  urine,  440,  676 

in  foetus,  411 

in  gall-bladder,  411,  675 

in  sputa,  411 
as  pus  producer,  410 
staining  characteristics  of,  403 
in  urine,  442 
infection  of,  by  contaminated 

drinking  water,  414 
by  milk,  414 
immunization  in,  415 

UREA,  fermentation  of,  67 
Urine,    bacteria   eliminated 
through,  117 


\J  ACCIN  ATION ,      immunity 

conferred  by,  649,  650 
Vaccine,  bacteria  in,  657 

bodies,  651 

durability  of,  656 

preparation  of,  652 

virus,  use  of,  650 
Vincent's  bacillus,  354 
Virulence,  how  diminished,  96 

how  increased,  96 

possessed  by  bacteria,  96 
Vitality,  increase  of,  strengthens 

immunity,  124 


WATER,  bacilli  of  colon  group 
in,  248 
bacteriological  examination  of, 

245 

contamination  and  purification 
of  drinking   253 


Water,  domestic  purification  of, 

255 
obtaining  of,  for  examination, 

249 

typhoid  bacilli  in,  247 
Whooping-cough,  bacillus  of,  613 
Widal  reaction,  421 
test,  419 

advantages  and  disadvan- 
tages of  serum  and  dried 
blood  in,  424 

dilution   of  blood- serum   to 

be  employed  and  time  re 

quired  for  development  of, 

reaction  of,  425 

mode  of  obtaining  serum  in, 

423 
persistence   of    reaction   in, 

428 

proportion  of  cases  in  which 
definite  reaction  occurs  in, 
427 

pseudo  reactions,  422 
reaction    with    blood-serum 
of  healthy  persons,  428 
of  those  ill  with  diseases 
other  than  typhoid  fever, 
428 

use  of  dried  blood  in,  420 
WolfFhiigel's  apparatus  for  count- 
ing colonies,  227 


VEROSIS  bacilli,  347 
A 


YEASTS,  625 

1      Yellow  fever,  bacillus  of,  609 


yiEHL'S    carbol-fuchsin    solu- 
L     tlon  for  tubercle  bacilli,  303 
carbolic  fuchsin  solution,  202, 
Ziehl-Neelson's  method,  310 


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